CN109517817B - Method for preparing unit DNA composition and method for preparing DNA link - Google Patents

Abstract

The invention provides a method for preparing a unit DNA composition and a method for preparing a DNA link. The method for preparing the unit DNA composition comprises the following steps: preparing a solution containing each of a plurality of unit DNAs to which an additional sequence is ligated; and a step of preparing each solution, measuring the concentration of the unit DNA in each solution in a state where the additional sequence is connected to the unit DNA, and sorting each solution based on the result of the measurement so that the number of moles of the unit DNA in each solution is close to the same. The method for producing a DNA conjugate comprises the steps of: a step of preparing a unit DNA composition, a step of preparing a vector DNA, a step of removing each additional sequence from the unit DNA to which the additional sequence is linked in the prepared solution with a restriction enzyme, and a step of linking the vector DNA and each unit DNA to each other after the removal step.

Description

Method for preparing unit DNA composition and method for preparing DNA link

The present application is a divisional application of the chinese invention patent application 201480073826.9 (PCT application No. PCT/JP 2014/073579), having the application date of 2014, 9 and 5, and having the invention name of "method for preparing a unit DNA composition and method for producing a DNA conjugate".

Technical Field

The present invention relates to a method for preparing a unit DNA composition and a method for preparing a DNA conjugate.

Background

In recent years, development of a DNA synthesis technique for constructing a long-chain DNA on a genome level has been actively carried out. As a DNA synthesis technique, a technique of assembling unit DNAs obtained by chemically synthesizing DNAs and amplifying them by a PCR method is known. However, it is known that mutations are randomly introduced into synthetic DNA in the synthetic process of chemically synthesizing DNA and the PCR method. Therefore, in the assembly of genes, it is necessary to perform procedures of confirming the sequence of DNA and selecting DNA having a desired sequence at all stages up to the last stage.

In order to confirm the base sequence, the base sequence is generally determined by an automated fluorescent sequencer using the dideoxy chain terminator method (Sanger method), and a base sequence having a continuous length of about 800 bases can be confirmed in 1-time base sequence determination by this method. When the base sequence of a unit DNA synthesized by chemical synthesis or PCR is to be confirmed before gene assembly, the time and cost can be reduced if the number of times of base sequence measurement is small. Therefore, short unit DNA synthesized by chemical synthesis or PCR method is preferable for gene assembly.

However, if the unit DNA for gene assembly is short, it is necessary to assemble many unit DNAs.

Conventionally, as one of methods for assembling a plurality of unit DNAs, a gene assembly method (OGAB method) using a plasmid transformation system of Bacillus subtilis has been known. For example, patent document 1 discloses a method for preparing plasmid DNA for transforming bacillus subtilis cells using the OGAB method.

In the OGAB method, a so-called multimeric plasmid (multimer plasmid) in which a plurality of plasmid units exist in 1 DNA molecule by homologous recombination between plasmid molecules is used. When the OGAB method is used, the DNA molecule for transformation does not need to be circular DNA, and transformation of a plasmid is possible as long as it is formed into a tandem repeat (tandem repeats) in which 1unit of a plasmid and each unit DNA for assembly are repeated in the same direction.

In the OGAB method, since it is necessary to prepare DNA molecules in which ligation (ligation) is repeated in tandem as described above, when a plurality of unit DNAs are used, it is necessary to join these unit DNAs to a plasmid. However, as the kinds of unit DNAs are increased, it becomes difficult to connect the unit DNAs to create tandem repeats, and it is desirable that the molar ratio of each unit DNA at the time of connection is close to 1 in order to connect a large number of unit DNAs.

Documents of the prior art

Patent document

Patent document 1: japanese patent No. 4479199

Disclosure of Invention

However, in practice, it is difficult to precisely control the number of moles of each unit DNA. One reason for this is that in the DNA measurement method using a fluorescent double-stranded DNA intercalator such as SYBRGeeni, the number of molecules can be measured with reproducibility of only about. + -. 20% due to extinction of a fluorescent substance during the measurement. In addition, in these measurement methods, the weight per unit volume of DNA is measured, and in the OGAB method, the unit DNA amount is calculated based on the number of moles per unit volume, so it is necessary to convert the measured DNA weight value obtained by the above measurement method into a molar concentration. Therefore, when the length distribution of the unit DNA molecules is wide, even if the number of moles of the unit DNA molecules is the same, the value calculated based on the measured value often contains a large error because the weight is proportional to the length of the unit DNA, and particularly, when the measured value of the weight differs by several times or more. In the method of patent document 1, although an attempt is made to adjust the molar ratio of each unit DNA to 1, the molar ratio cannot be precisely controlled because the length distribution of each unit DNA is wide.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for producing a unit DNA composition in which the number of moles of a plurality of unit DNAs is more uniform, and a method for producing a DNA construct.

The present inventors have found that the number of moles of unit DNAs is measured in a state where additional sequences are ligated to each unit DNA, thereby reducing the error of the measurement result, and have completed the present invention. More specifically, the present invention provides the following.

(1) A method of preparing a unitary DNA composition, the method comprising the steps of:

preparing a solution containing each of a plurality of unit DNAs to which an additional sequence is ligated; and

and a step of preparing each solution, measuring the concentration of the unit DNA in each solution in a state where the unit DNA is linked with an additional sequence, and sorting each solution based on the result of the measurement so that the number of moles of the unit DNA in each solution is approximately the same.

(2) The method for producing a unit DNA composition according to (1), wherein the unit DNA to which an additional sequence is ligated has a circular structure, and the additional sequence is a plasmid DNA sequence having an origin of replication.

(3) The method for producing a unit DNA composition according to (1) or (2), wherein a standard deviation of a distribution of total lengths of the respective base lengths of the unit DNAs and the total base length of an additional sequence linked to each unit DNA is within. + -. 20% relative to the average total length.

(4) The method for producing a unit DNA composition according to any one of (1) to (3), wherein the average base length of the additional sequence linked to each unit DNA is 2 times or more the average base length of the unit DNA.

(5) The method for producing a unit DNA composition according to any one of (1) to (4), wherein each of the unit DNAs has a length of 1600bp or less.

(6) The method for producing a unit DNA composition according to any one of (1) to (5), which is used for preparing a DNA conjugate containing an assembly DNA composed of the unit DNA,

in the method, the step of preparing a solution containing the unit DNA includes the steps of: the unit DNA having a non-palindromic sequence at an end thereof is designed so as to be bounded by non-palindromic sequences in the assembled DNA in the vicinity of the sequence at positions where the assembled DNA is equally divided into equal base lengths when the base length of the sequence of the assembled DNA is divided by the number of types of the unit DNA.

(7) A method for producing a DNA construct for use in microbial cell transformation, said DNA construct comprising more than 1 assembled DNA unit comprising a vector DNA and an assembled DNA, said vector DNA comprising an origin of replication effective in a host microorganism, said method comprising the steps of:

a step of preparing a unit DNA composition by the method according to any one of claims 1 to 6;

preparing the vector DNA;

removing each additional sequence from the prepared unit DNA having the additional sequence ligated to the solution using a restriction enzyme; and

a step of linking the vector DNA and the unit DNAs to each other after the removal step,

wherein the vector DNA and the unit DNAs have a structure capable of being repeatedly linked in an order of each other,

the assembly DNA includes DNAs in which the unit DNAs are linked to each other.

(8) The method for producing a DNA construct according to (7), which comprises: and a step of adjusting the variation coefficient of the concentrations of the vector DNA and the unit DNAs in the ligation step based on a relational expression between the yield of DNA fragments and the variation coefficient of the concentrations of the DNA fragments, the target ligation number being expressed by the product of the number of unit DNAs constituting an assembly unit and the number of assembly units.

(9) The method for producing a DNA construct according to (7) or (8), wherein the restriction enzyme is a type II restriction enzyme.

(10) The method for producing a DNA conjugate according to any one of (7) to (9), further comprising a step of mixing at least 2 types of the unit DNA-containing solutions in the prepared unit DNA-containing solution before the removing step.

(11) The method for producing a DNA conjugate according to any one of (7) to (10), further comprising a step of inactivating the restriction enzyme after the removal step and before the ligation step.

(12) The method for producing a DNA construct according to any one of (7) to (11), wherein the microorganism is Bacillus subtilis.

According to the present invention, a method for producing a single DNA composition and a method for producing a DNA assembly, in which the number of moles of a plurality of single DNAs is more uniform, can be provided.

Drawings

FIG. 1 is a diagram showing a vector DNA according to an embodiment of the present invention.

FIG. 2 is a diagram showing the structure of a synonymous codon mutant in the 10 th fragment in the unit DNA according to example 1 of the present invention.

FIG. 3 is a photograph showing the electrophoresis of a crude plasmid containing the 01. Sup. Th fragment or the 21. Sup. Th fragment of the unit DNA in example 1 of the present invention, and a high purity plasmid obtained by purifying the crude plasmid, which was treated with a restriction enzyme.

FIG. 4 shows electrophoretograms of all the treatment with various restriction enzymes for the group to be treated with BbsI, the group to be treated with AarI, and the group to be treated with BsmBI among the purified plasmids containing unit DNA in example 1 of the present invention.

FIG. 5 shows electrophoretograms after the restriction enzymes were all treated with various restriction enzymes and the groups were integrated for the group to be treated with BbsI, the group to be treated with AarI, and the group to be treated with BsmBI among the plasmids containing unit DNAs purified in example 1 of the present invention.

FIG. 6 is a graph showing the distribution of the number of molecules of each unit DNA before and after size fractionation (size fractionation) after all groups were integrated by treating the group to be treated with BbsI, the group to be treated with AarI, and the group to be treated with BsmBI with various restriction enzymes in a plasmid containing the unit DNA according to example 1 of the present invention.

FIG. 7 is a graph showing the rate of change in the number of molecules of each unit DNA before and after size fractionation, after all groups were treated with various restriction enzymes and integrated, with respect to the group to be treated with BbsI, the group to be treated with AarI, and the group to be treated with BsmBI, in the plasmid containing unit DNA in example 1 of the present invention.

FIG. 8 is a photograph showing electrophoresis after the unit DNA and the carrier DNA are ligated in example 1 of the present invention.

FIG. 9 is a photograph showing electrophoresis after a DNA conjugate obtained by ligating a unit DNA and a vector DNA in example 1 of the present invention was transformed into Bacillus subtilis, plasmids were extracted from the transformed Bacillus subtilis, and the plasmids were treated with a restriction enzyme.

FIG. 10 is a photograph showing the results of screening a clone of Bacillus subtilis containing the desired assembly DNA based on the results of electrophoresis after extracting plasmids from a plurality of transformed Bacillus subtilis after transformation and treating the plasmids with restriction enzymes, and electrophoresis after restriction enzyme treatment in example 1 of the present invention.

FIG. 11 shows that the selected assembly DNA forms plaques (plaques) of lambda phage DNA in example 1 of the present invention.

FIG. 12 is a photograph showing the genome of the selected assembled DNA and a wild type lambda phage after being treated with AvaI restriction enzyme in example 1 of the present invention.

FIG. 13 is a photograph showing electrophoresis after treating the whole of a purified plasmid containing a unit DNA with AarI restriction enzyme in example 2 of the present invention.

FIG. 14 is a photograph showing electrophoresis after ligation of unit DNA and carrier DNA in example 2 of the present invention.

FIG. 15 is a photograph showing electrophoresis after a DNA assembly obtained by ligating a unit DNA and a vector DNA is transformed into Bacillus subtilis, plasmids are extracted from the transformed Bacillus subtilis, and the plasmids are treated with a restriction enzyme in example 2 of the present invention.

FIG. 16 is a photograph showing the result of screening a clone of Bacillus subtilis containing the desired assembly DNA based on the result of electrophoresis after extracting plasmids from a plurality of transformed Bacillus subtilis after transformation and treating the plasmids with restriction enzymes, and electrophoresis after restriction enzyme treatment in example 2 of the present invention.

FIG. 17 shows photographs of electrophoresis of DNAs (A) to (H) used in test example 1.

FIG. 18 is a graph showing the number of transformants of Bacillus subtilis competent cells transformed with the DNAs (A) to (H) used in test example 1.

FIG. 19 is a graph showing the relationship between the difference CV (%) in the concentration of unit DNA fragments and the relative amount of each unit DNA fragment in each gene assembly scale of model 1. (a) is a graph for 6 fragment assembly, (b) is a graph for 13 fragment assembly, (c) is a graph for 26 fragment assembly, and (d) is a graph for 51 fragment assembly.

Fig. 20 is a graph showing the relationship between N (the number of unit DNAs contained in 1 ligation product) and the number of ligation product molecules when CV =20% in the case of simulating assembly of 6 fragments of 1.

In FIG. 21, (a) is a graph of a lambda function of the difference CV (%) in unit DNA fragment concentration derived by fitting to an exponential distribution curve in simulation 1, and (b) is a graph of a lambda function of the difference CV (%) in unit DNA fragment concentration obtained from an average of N values of the simulated ligated products.

FIG. 22 shows mislinkage sites (mislinkage sites) of #1, #2, #5, #7, #8, #9, #10 and #11 in the assemblies obtained in the experiment for lambda phage genome reconstruction in mock 1.

Fig. 23 shows photographs after pulsed field gel electrophoresis of the ligation products of 51 unitary DNA fragments with a CV =6.6% difference in the number of fragments in the experiment for lambda phage genome reconstruction in mock 1.

Fig. 24 is a graph comparing the actual connection efficiency and the connection efficiency obtained by the connection simulation in the simulation 1. In the figure, (a) is a graph compared with a simulation having a connection efficiency of 95%, (b) is a graph compared with a simulation having a connection efficiency of 96%, (c) is a graph compared with a simulation having a connection efficiency of 97%, (d) is a graph compared with a simulation having a connection efficiency of 98%, (e) is a graph compared with a simulation having a connection efficiency of 99%, and (f) is a graph compared with a simulation having a connection efficiency of 100%.

Fig. 25 is a graph showing the relationship between the difference CV (%) in the unit DNA concentration fragments and the relative amount of each unit DNA fragment in each gene assembly scale of model 1, obtained by the general formula f (N) =0.0058 × CV (%) -exp (-0.058 × CV (%) -N).

Fig. 26 is a graph showing the relationship between the difference in the concentration of the unit DNA fragments and the average number of unit DNA fragments of 1 ligation product, obtained by the general formula f (N) =0.0058 × cv (%) × exp (-0.0058 × cv (%) × N).

Detailed Description

Embodiments of the present invention will be described below, but the present invention is not limited to these examples.

< method for producing Unit DNA composition >

The method for preparing the unit DNA composition of the present invention comprises the following steps: preparing a solution containing each of a plurality of unit DNAs to which an additional sequence is ligated; and a step of preparing each solution, measuring the concentration of the unit DNA in each solution in a state where the unit DNA is linked with an additional sequence, and sorting each solution based on the result of the measurement so that the number of moles of the unit DNA in each solution is approximately the same. In the present specification, the type of "unit DNA" is distinguished according to the base sequence of each DNA. In addition, unit DNAs to which a restriction enzyme recognition site is attached and which are not attached are both included in the "unit DNA".

In the present invention, when the concentration of each unit DNA in a solution containing the unit DNA is measured, an additional sequence is ligated to each unit DNA. Thus, the length distribution of the base sequence decreases when the concentration of the solution is measured due to the additional sequence linked thereto. Therefore, the error in the number of moles of each unit DNA calculated based on the measurement result is reduced. Therefore, by dividing each solution based on the measurement result and adjusting the number of moles of the unit DNA in each solution to be the same, the molar ratio in each solution is easily close to 1. The "concentration of the unit DNA in the solution" measured above means the molar concentration of the unit DNA. The method for measuring the molar concentration of the unit DNA in the solution is not particularly limited, and for example, the mass% of the unit DNA in the solution may be measured, and the molar concentration of the unit DNA in the solution may be calculated from the value of the mass% of the unit DNA in the measured solution. The method for measuring the molar concentration of unit DNA in a solution is preferably a method capable of measuring with an accuracy within. + -. 20% by weight of the DNA, and more specifically, ultraviolet absorption spectroscopy using a microabsorption photometer is preferably used.

The step of preparing a solution containing the unit DNA to which the additional sequence is ligated is not particularly limited, and may be carried out, for example, by preparing the unit DNA and ligating the additional sequence to the unit DNA thereafter.

The preparation of the unit DNA may be performed by using a previously synthesized unit DNA, or may be performed by preparing the unit DNA. The unit DNA can be prepared by a conventionally known method, for example, by a chain polymerase reaction (PCR) or chemical synthesis. When a restriction enzyme recognition sequence is added to the unit DNA, it may be prepared by PCR using a primer to which a restriction enzyme recognition sequence is added (in which each protruding end is generated on the base sequence of the template DNA), or by chemical synthesis by previously incorporating a restriction enzyme recognition sequence so that an arbitrary protruding sequence can be generated at the end. The base sequence of the prepared unit DNA can be confirmed by a conventionally known method, for example, by incorporating the unit DNA into a plasmid and measuring the base sequence by an automated fluorescent sequencer using the dideoxy chain termination method.

The additional sequence is not particularly limited, and may be a linear DNA or a circular plasmid. When a circular plasmid DNA sequence is used, the unit DNA to which the additional sequence is ligated has a circular structure, and thus can be transformed into a host such as Escherichia coli.

The kind of plasmid DNA is not particularly limited, and in order to replicate the plasmid DNA in a transformed host, it is preferable that the plasmid DNA sequence has an origin of replication. In particular, the high copy plasmid vector pUC19 of Escherichia coli or a plasmid derived therefrom is preferable. In addition, from the viewpoint of reducing the length distribution between DNAs to which additional sequences are ligated, thereby making the number of moles of unit DNAs more similar to each other, it is preferable that all unit DNAs be cloned into the same kind of plasmid vector.

The additional sequence and the unit DNA may be ligated by, for example, ligation using DNA ligase, or in the case of ligation to plasmid DNA, they may be ligated by TA cloning.

The standard deviation of the distribution of the total length of the base lengths of the unit DNAs and the additional sequences linked to the unit DNAs is not particularly limited, and when the standard deviation is small, the error in the number of moles of each unit DNA calculated based on the measurement result of the concentration of DNA in a solution is reduced, and therefore, the number of moles of unit DNA in each solution can be made closer to the same as each other. Specifically, the standard deviation of the distribution of the total length of each base length of the unit DNA and the total base length of the additional sequence linked to each unit DNA is preferably within. + -. 20%, more preferably within. + -. 15%, even more preferably within. + -. 10%, even more preferably within. + -. 5%, even more preferably within. + -. 1%, and most preferably within. + -. 0.5% of the average total length.

The average base length of the additional sequence linked to each unit DNA is not particularly limited, and when it is longer than the average base length of the unit DNA, the error in the number of moles of each unit DNA calculated based on the measurement result of the concentration of DNA in the solution is reduced, and therefore, the number of moles of unit DNA in each solution can be made closer to the same as each other. Specifically, the average base length of the additional sequence linked to each unit DNA is preferably 2 times or more, more preferably 5 times or more, still more preferably 10 times or more, and most preferably 20 times or more, the average base length of the unit DNA. In addition, if the average base length of the additional sequence ligated to the unit DNA is too long, it will be difficult to manipulate the unit DNA ligated with the additional sequence. Therefore, the average base length of the additional sequence linked to each unit DNA is preferably 10000 times or less (specifically, 5000 times or less, 3000 times or less, 1000 times or less, 500 times or less, 250 times or less, 100 times or less, etc.) or the like, relative to the average base length of the unit DNA.

The length of each unit DNA is not particularly limited, but is preferably determined by determining the base sequence of the unit DNA, because if the number of times of base sequence determination is small, the time and cost can be reduced. Therefore, the length of each unit DNA is preferably short, specifically, 1600bp or less, and more preferably 1200bp or less. In particular, when the base sequence is determined by an automated fluorescent sequencer using the dideoxy chain termination method, a base sequence having a continuous length of about 800 bases can be confirmed 1 time of base sequence determination, and therefore, the length of each unit DNA is most preferably 800bp or less (specifically, 600bp or less, 500bp or less, 400bp or less, 200bp or less, 100bp or less, and the like). In this case, since each unit DNA is short in length, a large number of unit DNAs are required for preparing a DNA conjugate described below. However, if the unit DNA prepared by the method of the present invention is used, as described later, the ligation of a large number of unit DNAs can be achieved. In addition, if the length of each unit DNA is too short, the number of unit DNAs increases, and the operation efficiency decreases. Therefore, the length of each unit DNA is preferably 20bp or more, more preferably 30bp or more, and still more preferably 50bp or more.

The use of the unit DNA composition according to the preparation method of the present invention is not particularly limited, and the unit DNA composition prepared by the preparation method according to the method of the present invention can be used for preparing a DNA construct containing an assembly DNA composed of the unit DNA. When a DNA conjugate is prepared by the method described later using the unit DNA composition prepared by the preparation method according to the method of the present invention, a large number of unit DNAs (for example, 50 or more kinds) can be ligated. This is probably because the number of moles of each unit DNA in the unit DNA composition prepared by the preparation method according to the present invention is more accurately approximated to the same number.

In the present invention, the step of preparing a solution containing unit DNA may include a step of designing unit DNA. The design of the unit DNA is not particularly limited, and for example, when the unit DNA composition is used to prepare a DNA assembly containing an assembly DNA, the unit DNA having a non-palindromic sequence at its end may be designed with the non-palindromic sequence in the vicinity of the sequence of the assembly DNA at a position where the base lengths of the assembly DNA are equally divided by the number of types of the unit DNA. When the unit DNAs are designed in the above manner, the lengths of the unit DNAs are substantially the same. Therefore, when the DNA is used for the preparation of a DNA aggregate described below, a band having substantially the same position appears in size fractionation after electrophoresis by removing an additional sequence with a restriction enzyme, and therefore, it is preferable from the viewpoint of improving the operation efficiency that the unit DNA can be recovered by 1 size fractionation. The "vicinity of the sequence of the position where the DNA is to be assembled and the like" is not particularly limited, and may be appropriately set according to the length of the base sequence. For example, when the base length of each unit DNA is 1000bp, it is possible that the base length is within 100bp (specifically, within 90bp, within 80bp, within 70bp, within 60bp, within 50bp, within 30bp, within 20bp, within 10bp, within 5bp, or the like) from the "position at which the assembled DNA is equally divided".

In the case of designing a unit DNA as described above, it is preferable to design a DNA linker containing a target assembly DNA such that the end of the unit DNA has a non-palindromic sequence (a sequence other than a palindromic sequence). When the above-described non-palindromic sequence is used as a protruding sequence, the unit DNA is designed so that the non-palindromic sequence can be repeatedly linked in an order-retaining state as described later.

< method for producing DNA construct >

The present invention includes a method for producing a DNA conjugate. According to the above method, the method for producing a DNA conjugate of the present invention comprises the steps of: a step of preparing a unit DNA composition, a step of preparing a vector DNA, a step of removing each additional sequence from the unit DNA to which the additional sequence is linked in the prepared solution with a restriction enzyme, and a step of linking the vector DNA and each unit DNA to each other after the removal step.

DNA ligations contain more than 1 assembled DNA unit (unit) for microbial cell transformation. The assembly DNA unit comprises a vector DNA and an assembly DNA. The number of DNA units assembled in the DNA construct is not particularly limited as long as it is greater than 1, but is preferably 1.5 or more, more preferably 2 or more, still more preferably 3 or more, and most preferably 4 or more, in order to improve the transformation efficiency.

The vector DNA has an origin of replication effective in the host microorganism to be transformed. The vector DNA is not particularly limited as long as it has a sequence that allows DNA replication in a microorganism capable of transforming the DNA construct, and examples thereof include sequences of replication origins effective in the bacteria of the genus Bacillus (Bacillus), which will be described later. The sequence of the replication origin effective in Bacillus subtilis is not particularly limited, and examples of the sequence having the replication mechanism of the theta-type include sequences of the replication origin contained in plasmids such as pTB19 (Imanaka, T., et al. J. Gen. Microbioli.130, 1399-1408 (1984)), pLS32 (Tanaka, T and Ogra, M.FEBS Lett.422, 243-246 (1998)), pAM β 1 (Swinfield, T.J., et al. Gene87, 79-90 (1990)), and the like.

The assembly DNA includes DNAs in which the above-described unit DNAs are linked to each other. The DNA in the present invention is a DNA to be cloned, and the type and size thereof are not particularly limited. Specifically, the sequence may be a naturally-derived sequence such as a prokaryote, a eukaryote, or a virus, or an artificially designed sequence. In the method of the present invention, since a large number of unit DNAs can be ligated to a plasmid as described above, it is preferable to use a DNA having a long base length. Examples of the DNA having a long base length include the whole of genomic DNA of a gene group, phage, or the like constituting a series of metabolic pathways, and a part of genomic DNA.

The assembly DNA may or may not contain an appropriate base sequence, as required, in addition to the vector DNA and the assembly DNA. For example, a plasmid for expressing a gene contained in an assembly DNA may be prepared to contain a nucleotide sequence for controlling transcription and translation, such as a promoter (promoter), an operator (operator), an activator (activator), and a terminator (terminator). Specific examples of the promoter in the case of using Bacillus subtilis as a host include Pspac (Yansura, D.and Henner, D.J.Pro.Natl.Acad.Sci, USA 81, 439-443. (1984.)) and Pr promoter (Itaya, M.biosci.Biotechnol.biochem.63, 602-604. (1999)) which can control expression using IPTG (isopropyl-. Beta. -D-thiogalactoside, isopyropyranoside).

The vector DNA and each unit DNA have a structure capable of being repeatedly linked in a state of maintaining the order of each other. In the present specification, the phrase "linked while maintaining the order of each other" means that unit DNAs or vector DNAs having sequences adjacent to each other in an assembled DNA unit are bound while maintaining the order and orientation thereof. The term "repetitive linkage" means that the 5 '-end of the unit DNA or vector DNA having a base sequence at the 5' -end is linked to the 3 '-end of the unit DNA or vector DNA having a base sequence at the 3' -end. Specific examples of such a unit DNA include a unit DNA having ends which can be repeatedly linked in a state of maintaining the order of the ends of the fragments by utilizing the complementarity of the nucleotide sequences of the protruding ends of the fragments. The structure of the overhang is not particularly limited, and when it is a non-palindromic sequence, the shape of the 5 '-end overhang may be different from that of the 3' -end overhang.

The overhang ends are preferably prepared by a step of removing each additional sequence from the unit DNA using a restriction enzyme. Therefore, when a DNA construct is prepared by the method of the present invention, the unit DNA preferably has a restriction enzyme recognition sequence so that the added sequence can be removed by the restriction enzyme. For the preparation of the vector DNA, protruding ends capable of allowing the vector DNA and the unit DNA to be repeatedly ligated in a state of maintaining the order of each other can be provided by, for example, performing a restriction enzyme treatment.

The restriction enzyme used for removing the additional sequence is not particularly limited, but is preferably a type II restriction enzyme, and more preferably a type IIS restriction enzyme capable of forming an overhanging end of an arbitrary sequence at a position having a fixed length from the outside of the recognition sequence, such as AarI, bbsI, bbvI, bcoDI, bfuAI, bsaI, bsaXI, bsmAI, bsmBI, bsmFI, bspMI, bspQI, btgZI, fokI, or SfaNI. When the type IIS restriction enzyme is used, the protruding ends of the unit DNAs can be made different at each ligation site, and thus the ligation order can be secured. In addition, in preparation of the vector DNA, similarly to the preparation of the unit DNA, it is preferable to prepare the vector DNA with the type IIS restriction enzyme so as to provide a protruding end capable of repeatedly linking with the unit DNA in a state of maintaining the order of the unit DNA.

When the unit DNAs are classified into groups according to the type of restriction enzyme used for removal of the additional sequence, 2 or more kinds of solutions containing the unit DNAs may be mixed for each group before the removal step. Thus, it is not necessary to perform restriction enzyme treatment on each unit DNA, but restriction enzyme treatment can be performed on each restriction enzyme group at a time, and when separation of electrophoresis unit DNA is performed, for example, unit DNA can be recovered by separation at a time, so that the efficiency of the operation is improved. In addition, when a plurality of unit DNA groups are present, when separation and recovery of the unit DNA are performed for each group, an error in the amount of recovery may occur between the groups, and therefore, an error may occur in the number of moles unified among the unit DNAs. Therefore, when the restriction enzymes used for removal of the additional sequence are classified into groups, it is preferable that the number of groups is small, that is, it is preferable that the number of restriction enzymes used for removal of the additional sequence is small. Therefore, the types of restriction enzymes used are preferably 5 or less, more preferably 3 or less, and most preferably 1. That is, if one restriction enzyme is used, all the solutions containing the unit DNAs can be mixed, and therefore, the efficiency of the operation is greatly improved, and errors are not easily caused in the number of moles unified among the unit DNAs. Since restriction enzymes are mixed in a state in which the number of moles is substantially equal, a large number of unit DNAs can be ligated even when such a mixed solution is used.

When the type IIS restriction enzyme recognition sequence is added to the unit DNA, the restriction enzyme recognition sequence of the unit DNA is designed so as not to recognize the sequence possessed by each unit DNA. That is, the restriction enzyme recognition sequences of the respective unit DNAs were designed as follows: when it is intended to use a type IIS restriction enzyme, if the type IIS restriction enzyme does not recognize the sequence of a unit DNA but recognizes the sequence of another unit DNA, a type IIS restriction enzyme different from the type IIS restriction enzyme is used for the other unit DNA. In this manner, although the type IIS restriction enzyme used for each unit DNA is different, the unit DNA can be used by classifying it into the above-mentioned groups according to the type of the type IIS restriction enzyme. If there is a type IIS restriction enzyme that does not recognize any of the sequences in each unit DNA, it is possible to remove additional sequences from all the unit DNAs using 1 type IIS restriction enzyme by designing the unit DNA to which the recognition sequence of the restriction enzyme is added.

The step of ligating the vector DNA and each unit DNA to each other is not particularly limited, and may be carried out by subjecting the vector DNA and each unit DNA to a restriction enzyme treatment, separating the addition sequence and the unit DNA after the restriction enzyme treatment, and ligating (ligating) the separated unit DNA with the vector DNA by using a DNA ligase or the like. Thus, a DNA construct for transforming a microorganism can be prepared. The unit DNA in the ligation step is a unit DNA to which a restriction enzyme recognition sequence is not added.

The method for separating the additional sequence and the unit DNA is not particularly limited, and a method in which the relationship between the molar ratio of each unit DNA after the restriction enzyme treatment does not change significantly is preferable, and specifically, it is preferable to use agar gel electrophoresis.

The method for linking the unit DNA and the vector DNA is not particularly limited, and it is preferably carried out in the presence of polyethylene glycol and a salt. The salt is more preferably a salt of an alkali metal having a valence of 1. More specifically, it is more preferable to carry out the ligation reaction in a ligation reaction solution containing 10% of polyethylene glycol 6000 and 250mM of sodium chloride. The concentration of each unit DNA in the ligation reaction solution is not particularly limited, but is preferably 1 fmol/. Mu.l or more. The temperature and time of the ligation reaction are not particularly limited, but are preferably 37 ℃ for 30 minutes or more. In addition, it is preferable that the concentration of the DNA in the solution containing the carrier DNA is measured before the reaction and is adjusted to be equal to the number of moles of the unit DNA in the ligation reaction solution.

The method for preparing a DNA conjugate according to the present invention may or may not include (but preferably includes) the following steps: the variation coefficient of the concentrations of the vector DNA and each unit DNA in the ligation step (hereinafter referred to as "variation coefficient 2" in the present specification) is adjusted based on a relational expression (hereinafter referred to as "relational expression" in the present specification) between the yield of DNA fragments of the target ligation number (expressed by the product of the number of DNA units constituting the assembly unit and the number of assembly units) and the variation coefficient of the concentration of the DNA fragments (hereinafter referred to as "variation coefficient 1" in the present specification). Note that the variation coefficient 1 is a variation coefficient used for convenience in the relational expression, and the variation coefficient 2 is a variation coefficient of the concentrations of the unit DNA and the carrier DNA in the actual linking step. By including this adjustment step, when an attempt is made to join a desired number of unit DNAs (for example, unit DNAs having a fragment number of 50) in the joining step, such joining can be achieved by adjusting the variation coefficient 2 to the range shown in the above relational expression.

The target number of linked DNA fragments is the number of desired DNA fragments to be linked in the linking step, and more specifically, is expressed by multiplying the number of unit DNAs constituting the linked assembly units by the number of assembly units. The "yield of DNA fragments of a target binding number" refers to a ratio of the number of fragments of DNA constituting the assembled unit DNA after binding to the total number of fragments of DNA used for binding.

The relational expression according to the present invention is a relational expression showing the relationship between the yield of the target number of DNA fragments and the variation coefficient 1, and for example, a relational expression obtained by computer simulation (ligation simulation) can be used. More specifically, the relational expression may be set by the following method: for example, a ligation simulation is performed on a population of unit DNA fragments (for example, 10 to 30 populations) in which the variation coefficient 1 is 0 to 20% and which varies in units of 1%, the distribution (for example, exponential distribution) of the number of unit DNA fragments of a DNA assembly is examined, a fitting curve (fitting curve) is drawn for the distribution, and the setting is performed using the fitting curve. The tool used for the specific connection simulation is not particularly limited, and a conventional known means may be used, and for example, the simulation may be performed by programming VBA (Visual Basic for Applications) of table computing software Excel (registered trademark) 2007 and setting a necessary algorithm. In addition, the fitting curve may be plotted using an exponential approximation curve function such as tabular calculation software Excel (registered trademark) 2007. The adjustment of the variation coefficient 2 based on the relational expression can be performed as follows: for example, after designing the relational expression, the operation in each step before ligation is adjusted so that the DNA fragments at the time of ligation satisfy the calculated variation coefficient 1 by substituting the yield of the desired DNA fragment into the relational expression. The adjustment method is not particularly limited, and for example, in the case of measuring the concentration of the carrier DNA or each unit DNA in each step such as the step of preparing the carrier DNA, the step of preparing the unit DNA, and the step of connecting the carrier DNA and each unit DNA, when a measuring device (an absorptiometer, a real-time PCR device, or the like) used for the measurement is selected, a device for which the measurement error is determined in advance may be selected so that the variation coefficient 2 becomes a desired variation coefficient.

The variation coefficient 2 is not particularly limited, and if the error in the concentration of the unit DNA during the ligation is small, more unit DNAs can be ligated. Therefore, the coefficient of variation 2 is preferably 20% or less, more preferably 15% or less, further preferably 10% or less, further preferably 8% or less, and most preferably 5% or less.

The method for preparing a DNA conjugate of the present invention may further comprise a step of inactivating the restriction enzyme after the removal step and before the ligation step. If a restriction enzyme cleavage site for cleaving an additional sequence for separating other unit DNAs is present in the unit DNA, it is difficult to mix the unit DNA groups having the additional sequence at a stage before the inactivation of the restriction enzyme. Therefore, it is impossible to separate the unit DNAs at once by integrating these unit DNA groups. However, by inactivating the restriction enzyme, a group of unit DNAs having additional sequences can be integrated after inactivation, and the unit DNAs can be isolated together. Thus, in the ligation step, a DNA concatemer containing a larger number of assembled DNA units can be easily prepared, and as a result, transformation of Bacillus subtilis can be more easily performed. The inactivation of the restriction enzyme can be carried out by a conventionally known method, for example, by phenol/chloroform treatment.

The host microorganism to be transformed is not particularly limited as long as it is a microorganism having a natural transformation ability. Examples of the natural transformation ability include an ability to convert a DNA into a single-stranded DNA when introducing the DNA, and introduce the DNA. Specifically, there may be mentioned bacteria belonging to the genus Bacillus, bacteria belonging to the genus Streptococcus, bacteria belonging to the genus Haemophilus (Haemophilus), and bacteria belonging to the genus Neisseria (Neisseria). Examples of the bacillus bacteria include b.subtilis, b.megaterium, and b.stearothermophilus. Among these, the most preferred microorganism is Bacillus subtilis which is excellent in natural transformation ability and recombination ability.

The DNA construct prepared by the method of the present invention can be used for transformation of microbial cells, and a known method suitable for each microorganism can be selected as a method for making a microorganism to be transformed competent. Specifically, for example, in the case of Bacillus subtilis, the method described in Anagnostopoulou, C.and Spizzen, J.J.Bacteriol.,81, 741-746 (1961) is preferably used. Further, as for the method of transformation, known methods suitable for each microorganism may also be used. The amount of the ligation product solution to be supplied to competent cells is also not particularly limited. Preferably 1/20 to the same amount, more preferably half the amount, relative to the culture broth of competent cells. The method for purifying a plasmid from a transformant can also use a known method.

Whether or not the plasmid purified from the transformant contains the assembled DNA can be confirmed by the size pattern (size pattern) of the fragment generated by restriction enzyme cleavage, the PCR method, or the nucleotide sequence determination method. In addition, when the inserted DNA has a function of producing a substance or the like, it can be confirmed by detecting the function.

Examples

The present invention will be described in more detail with reference to the following examples, but the following examples are merely illustrative of the present invention and the scope of the present invention is not limited to the following examples.

(Material)

Bacillus subtilis is used as a microbial cell to be transformed. As bacillus subtilis, RM125 strain (Uozumi, t., et al, moi. Gen. Genet.,152, 65-69 (1977)) and a derivative strain BUSY9797 strain thereof were used. As vector DNA replicable in Bacillus subtilis, pGETS118-AarI-pBR (see sequence No. 31) and pGETS151-pBR (see sequence No. 32) constructed as described later using pGET118 (Kaneko, S., et al. Nucleic Acids Res.31, e112 (2003)) were used. As the assembly DNA, lambda phage DNA (manufactured by Toyo Boseki Co., ltd.) (see SEQ ID NO: 33) and a mevalonate pathway artificial operon (operon) described later (see SEQ ID NO: 34) were used. For E.coli screening with plasmid DNA having unit DNA integrated therein, carbenicillin (Wako pure chemical industries, ltd.) was used as an antibiotic. For the screening of Bacillus subtilis, antibiotic tetracycline (Sigma) was used. As the type IIS restriction enzyme, aarI (Thermo), bbsI (NEB), bsmBI (NEB) and SfiI (NEB) were used. Restriction enzymes HindIII, pvuII and T4DNA Ligase manufactured by Takara Bio were used. A usual Ligation for plasmid construction of E.coli was performed using Takara light Kit (Mighty) (Takara Bio Inc.). KOD plus polymerase from Toyo Boseki was used for PCR for preparing unit DNA. For colony PCR (colony PCR) for determining the nucleotide sequence of the DNA cloned into the plasmid, ex-Taq HS manufactured by Takara Bio Inc. was used. pMD-19 (simple) (Takara Bio Inc.) was used as plasmid DNA to which an additional sequence of the integration unit DNA was added. The circular Plasmid purification enzyme Plasmid Saf e used EPICENTER. As the agar gel for electrophoresis, 2-Hydroxyyethyagalose (Sigma) or UltraPure Agarose (Invitrogen) which is a low melting point agar gel used for DNA electrophoresis was used. A mixture of phenol, chloroform and isoamyl alcohol 25: 24: 1 was used for inactivation of restriction enzymes, and a reagent manufactured by Nacalai Tesque was used for TE-saturated phenol (containing 8-hydroxyquinoline). As the lambda-terminal enzyme, an enzyme manufactured by EPICENTER was used. Packaging of lambda phage (Packaging) Gigapack III Plus Packaging Extract from Agilent Technologies was used. The lysozyme was an enzyme manufactured by Wako pure chemical industries, ltd. As the medium components and agar for LB medium, a reagent manufactured by Becton, dickinson and Company was used. IPTG (isopropyl-. Beta. -D-thiogalactopyranoside) A reagent manufactured by Wako pure chemical industries, ltd. Was used. All the media components and biochemical reagents other than those described above were those manufactured by Wako pure chemical industries, ltd. Unless otherwise specified, E.coli DH 5. Alpha. Strain, JM109 strain or TOP10 strain was used for the construction of the plasmid. The constructed plasmid was purified from E.coli in a small amount using QIAprep Spin Miniprep Kit from QIAGEN, and in a large amount using QIAfilter Midi Kit from the same company. The MinElute Reaction Cleanup Kit from QIAGEN, or the QIAquick PCR purification Kit from QIAGEN was used for the purification and collection of DNA from the enzyme Reaction mixture. For purification of the Gel block separated by the usual agar Gel electrophoresis, minElute Gel Extraction Kit from QIAGEN was used. A nano-drop2000 available from Thermo was used as a microabsorption photometer. For the nucleotide sequence determination, a fluorescence automatic sequencer 3130xl Genetic Analyzer manufactured by Applied Biosystems was used. For other common DNA manipulations, standard protocols were followed (Sambrook, J., et al., molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, cold Spring Harbor, new York (1989)). Transformation of Bacillus subtilis and plasmid extraction were carried out according to known methods (Tsuge, K., et al., nucleic Acids Res.31, e133. (2003)).

(construction of vector DNA for Assembly)

The vector DNA for lambda phage DNA assembly, pGETS118-AarI-pBR (seq id No. 1), is a plasmid constructed by a multi-stage process based on an escherichia coli-bacillus subtilis shuttle (shuttle) plasmid vector pGETS118 (Kaneko, et al., nucleic Acids res.,31, e112. (2003)) having an origin of replication oriS of F factor of escherichia coli and an origin of replication repA that functions in bacillus subtilis, and its structure is shown in fig. 1. The cloning site of the assembled gene is located between 2 AarI cleavage sites, and the sequences between these 2 AarI cleavage sites are removed during assembly, and the origin of replication of E.coli multicopy plasmid pBR322 and the ampicillin resistance gene are introduced to make it easier to obtain the vector in E.coli. In addition, the recognition site was deleted by introducing a 1-base mutation into the natural AarI cleavage site present in the tetracycline resistance gene in pGETS118 so as not to affect the amino acid sequence of the tetracycline resistance gene (tetL). pGETS151-pBR (SEQ ID NO: 2), which is a vector DNA used for assembling a mevalonate pathway artificial operon, is a vector DNA prepared by connecting fragments amplified by PartA (5'-TAGGGTCTCAaagcggccgcaagctt-3' (see SEQ ID NO: 5) and 5'-TAGGGTCTCAGCggccaagaaggcc-3' (see SEQ ID NO: 6)), partB (5'-TAGGGTCTCAccGCCCTTCCCGGTCGATAT-3' (see SEQ ID NO: 7) and 5'-TAGGGTCTCAtaTTAGCTTAATTGTTATCCGCTCACAATTCC-3' (see SEQ ID NO: 8)), and PartC (5'-TAGGGTCTCAAAtaactggaaaaaattagtgtctcatggttcg-3' (see SEQ ID NO: 9) and 5'-TAGGGTCTCAgcttaagtggtgggtagttgacc-3' (see SEQ ID NO: 10)) using the above-described DNA of pGETS118-AarI-pBR as a template, and gene regions (gene regions between cat-oriS and between par A-parC) that act only in Escherichia coli are removed as compared with the original plasmid (FIG. 1). This vector DNA was able to replicate only in Bacillus subtilis during gene assembly, but exhibited the same properties as pGETS118-AarI-pBR during gene assembly. To about 10. Mu.l of a solution of these plasmids (equivalent to 5. Mu.g), 29. Mu.l of sterile water, 5. Mu.l of 10 Xbuffer _ for _ AarI attached to the restriction enzyme, 1. Mu.l of 50 XOligonletide for activating cleavage also attached to the restriction enzyme, and 5. Mu.l of restriction enzyme AarI (Thermo Co.) were added and reacted at 37 ℃ for 2 hours. After separating the resulting liquid by low-melting-point agar gel electrophoresis, a fragment of about 15kb (in the case of pGETS 118-AarI-pBR) or a fragment of 4.3kb (in the case of pGETS 151-pBR) of the vector itself was excised from the gel, and the DNA of the objective vector was purified and dissolved in 20. Mu.l of TE. For the measurement of the concentration of the carrier DNA, 1. Mu.l of the TE solution was taken and measured by an ultramicro absorptiometer.

(setting of Unit DNA dividing regions)

The diversity of the 4-base overhangs is 256 kinds, 4 th power of 4. The salient sequences used in the present invention were selected from them according to the following criteria. First, a total of 16 sequences (group 0) forming a palindrome (AATT, ATAT, TATA, TTAA, CCGG, CGCG, GCGC, GGCC, ACGT, AGCT, TCGA, TGCA, CATG, CTAG, GATC, GTAC) were subtracted, and complementary sequences of these sequences were also identical sequences, and the same fragments could be ligated to each other, and thus, could not be used in the present invention. Since the remaining 240 sequences include a certain sequence (e.g., CCTA) and a complementary sequence (TAGG) thereof, the combination of overhang sequences that can be used for DNA ligation is theoretically 240 ÷ 2=120 groups. Among them, the prominent combinations were grouped based on the following criteria according to the difference in GC content and the difference in the appearance order of these GC bases.

(group I) consists of A and T alone, for a total of 6 combinations (AAAA/TTTT, TAAA/TTTA, ATAA/TTAT, AATA/TATT, AAAT/ATTT, ATTA/TAAT).

(group II) A and T totals 3, and C and G totals 1, 32 combinations (CAAA/TTTG, ACAA/TTGT, AACA/TGTT, AAAC/GTTT, GAAA/TTTC, AGAA/TTCT, AAGA/TCTT, AAAG/CTTT, CAAT/ATTG, ACAT/ATGT, AACT/AGTT, AATC/GATT, GAAT/ATTC, AGAT/ATCT, AAGT/ACTT, AATG/CATT, CATA/TATG, ACTA/TAGT, ATCA/TGAT, ATAC/GTAT, GATA/TATC, AGTA/TACT, ATGA/TCAT, ATAG/CTAT, CTTA/TAAG, TCTA/TAGA, TTCA/TGAA, TTAC/GTAA, GTTA/TAAC, TGTA/TACA, TTGA/TCAA, TTCTAA/CTAA).

(group III) A and T totaled 2 and C and G totaled 2, 52 combinations from which 8 palindromic combinations were removed, 44 combinations (AACC/GGTT, AACG/CGTT, AAGC/GCTT, AAGG/CCTT, ACAC/GTGT, ACAG/CTGT, ACCA/TGGT, ACCT/AGGT, ACGA/TCGT, ACTC/GAGT, ACTG/CAGT, AGAC/GTCT, AGAG/CTCT, AGCA/TGCT, AGGA/TCCT, AGTC/GACT, AGTG/CACT, ATCC/GGAT, ATCG/CGAT, ATGC/GCAT, ATGG/CCAT, CAAC/GTTG, CAAG/CTTG, CACA/TGTG, CAGA/TCTG, CATC/GATG, CCAA/TTGG, CCTA/TAGG, CGAA/TTCG, CGTA/TACG, CTAC/GTAG, CTCA/TGAG, CTGA/TCAG, CTTC/GAAG, GAAC/GTTC, GACA/TGTC, GAGA/TCTC, GCAA/TTGC, GCTA/TAGC, GGAA/TTCC, GGTA/TACC, GTCA/TGAC, GTGA/TCAC, TCCA/TGGA).

(group IV) A and T were 1 in total and C and G were 3 in total, and 16 combinations in total without C and G triad (CACC/GGTG, CCAC/GTGG, CTCC/GGAG, CCTC/GAGG, CACG/CGTG, CCAG/CTGG, CTCG/CGAG, CCTG/CAGG, CAGC/GCTG, CGAC/GTCG, CTGC/GCAG, CGTC/GACG, GAGC/GCTC, GGAC/GTCC, GTGC/GCAC, GGTC/GACC) were used.

(group V) A and T are 1 in total, and C and G are 3 in total, and 16 combinations of C and G triads (ACCC/GGGT, CCCA/TGGG, TCCC/GGGA, CCCT/AGGG, ACCG/CGGT, CCGA/TCGG, TCCG/CGGA, CCGT/ACGG, ACGC/GCGT, CGCA/TGCG, TCGC/GCGA, CGCT/AGCG, AGGC/GCCT, GGCA/TGCC, TGGC/GCCA, GGCT/AGCC) are present.

(group VI) the protrusions consist of C and G only, in total 6 combinations (CCCC/GGGG, GCCC/GGGC, CGCC/GGCG, CCGC/GCGG, CCCG/CGGG, CGGC/GCCG).

Among the groups divided as described above, in examples 1 and 2, the boundary between the vector DNA and the unit DNA was selected from group 1. In addition, for the boundaries between the unit DNAs, 60 prominent combinations in total, i.e., group III (44 combinations) and group IV (16 combinations), were selected as candidate combinations. For the specification of each overhang pattern, the entire base sequence of the sequence to be assembled is first determined, and then ideal division boundaries for equally dividing the full-length sequence are set. The following describes a nucleotide sequence used in example 1 as a specific example.

In example 1, 48522bp, in which 16bp cos sites and 4bp overhangs necessary for assembly were added to 48502bp of the lambda phage genome in total, was used as a target for reconstitution. Table 1 below shows the ideal cleavage units, actual cleavage units and the protruding base sequences of the assembled DNAs in example 1. The unit DNA except for the assembly plasmid vector was divided into 50 pieces of almost the same size, and preparation was performed, and reconstruction by ligating these pieces was attempted. Ideally, it is desirable to divide a total of 50 fragments into fragments having equal lengths, but in order to not introduce any base change into the sequence to be assembled, it is necessary to create a 4-base 5' -end overhang for assembly based on the sequence originally present. However, the possibility that the above-mentioned overhang sequence is present exactly at all ideal division boundaries corresponds to 0, and thus the unit DNA cannot be equally divided at the ideal division boundaries. In this embodiment, in order to make the segment length as close as possible to the length of an ideal segmentation unit, a simulation was performed for assignment of a salient combination. First, 970bp obtained by dividing the total length (48522 bp) by 50 was used as an ideal segmentation unit, and the ideal segmentation unit was named as a 01 th fragment, a 02 th fragment, a 03 th fragment, a... Multidot.a.50 th fragment in this order from a unit DNA of a region having a small absolute base number. From the absolute positions of the ideal division boundaries (i.e., between 970 th and 971 th bases, 1940 th and 1941 th bases, 2910 th and 2911 th bases,.. 9., 47530, and 47531 bases), the positions were enlarged to 4 bases, 6 bases, 8 bases, 10 bases, 12 bases, 14 bases, 16 bases, 18 bases, 20 bases, 22 bases, and 24 bases at intervals of 1 base on the left and right sides of the ideal division boundaries, respectively, and whether or not there was a 4-base sequence that would be the above-mentioned alternative protrusion was examined. A specific example is described (table 1). The ideal split boundary between fragment 01 and fragment 02 is between bases 970 and 971. For 16 bases (5'-ATGCTGCTGGGTGTTT-3', which is a base sequence from the 963 rd base to the 988 th base) centered on this ideal division boundary, there are 7 kinds of the above-described alternative combinations (ACAC/GTGT, AGCA/TGCT, ATGC/GCAT, CACC/GGTG, CAGC/GCTG, CCAG/CTGG, CTGC/GCAG). By this operation, base sequences surrounding 49 ideal divided units in total were selected at a predetermined base distance, and it was confirmed whether or not at least 1 candidate overhanging sequence was present inside the base sequences. As a result, it was confirmed that if the selected base distance was extended to 24bp, at least 1 set of 4 bases highlighted as candidates existed in all the selected base sequences. When a specific protrusion is selected from among the candidate protrusions existing in each sequence, first, as described above, a unique protrusion combination is assigned to all the divided units by giving priority to the selected sequence having a small number of candidate protrusion combinations and giving priority to the protrusion combination sequence having a low frequency of appearance among all the selected sequences.

[ TABLE 1 ]

Example 1 preparation of Point mutants of lambda phage Using 50 Unit DNAs and vector DNAs

Lambda phage >

Lambda phages are bacterial phages (bacterial phages) that infect e.coli and are the most extensively studied phages in molecular biology. The genome consists of a double-stranded DNA of 48502bp in length, and all the nucleotide sequences have been identified. In addition, various mutants are known to exist. In this example, it was attempted to prepare a lambda phage spot mutant using a short unit DNA of about 1 kb.

Fragmentation design of lambda phage genome >

Lambda phagemid was produced by Toyo BoseDNA. The product was made linear at cos sites (sticky end sites). The full-length nucleotide sequence of the phage genome was investigated, and as a result, 6 differences (g.138delG, g.14266-14267insG, g.37589C > T, g.37743C > T, g.43082G > A, g.45352G > A) were observed in comparison with the nucleotide sequence registered in the database (accession No. J02459.1) (SEQ ID NO: 3) (the full-length of SEQ ID NO: 3 further contained 4 nucleotides of one overhanging end on the basis of the aforementioned 48522bp, that is, the full-length was 48526 bp). Using the obtained nucleotide sequences, in order to divide the total length of 48522bp (including the repetition of cos sites) into almost equal lengths, the ideal division boundaries were set to 970bp per fragment, and the fragments were divided by the above-described (method for setting the divided regions of the unit DNAs), and as a result, 5' -end overhangs composed of the right 4 bases of the cleavage sites shown in Table 1 were specifically assigned to each of the unit DNA groups.

< selection of the type of restriction enzyme producing the overhang >

Examples of the type IIS restriction enzymes that produce an arbitrary overhang sequence of 4 nucleotides include AarI (5 '-CACCTGC (N) 4/-3',5 '-/(N) 8 GCAGGTG-3'), bbsI (5 '-GAAGAC (N) 2/-3',5 '-/(N) 6 GTCTTC-3'), bbvI (5 '-GCAGC (N) 8/-3',5 '-/(N) 12 GCTGC-3'), bcoDI (5 '-GTCTCN/-3',5 '-/(N) 5 GAC-3'), bfuAI (5 '-ACCTGC (N) 4/-3',5 '-/(N) 8 GCAGGT-3'), bsaI (5 '-GGTCTCN/-3',5 '-/(N) 5 GAGACC-3'), bsmAI (isodiclomer of BcoDI), bsmBI (5 '-CGTCN/-3', 5'- (N) 5 GAGACG-3'), bsmFI (5 '-GGGAC (N) 10/-3',5 '-/(N) 14 GTCCC-3'), bspMI (isodicyme of BfuAI), btgI (5 '-GCGATG (N) 10/-3',5 '-/(N) 14 CATCGC-3'), fokI (5 '-GGATG (N) 9/-8978 zx8978' -/(N) 13CATCC-5 '), btgiC-3'), sfaNI (5 '-GCATC (N) 9/-3',5 '-/(N) 13 GATGC-5'), and the like. Among these restriction enzymes, those which do not exist in the E.coli plasmid vector (pMD 19, simple, TAKARA) for subcloning gene fragments or which, if present, produce fragments significantly larger and significantly smaller than the ideal cleavage unit are investigated, and as a result, there are 5 enzymes (AarI, bbsI, bfuAI, bsmFI, btgZI) which do not have their cleavage sites at all and 1 restriction enzyme (BsmBI) which, although a recognition sequence is present inside the vector, produce fragments significantly larger and significantly smaller than the ideal cleavage unit, i.e., 6 alternatives in total. The distribution of restriction enzyme sites was investigated for the whole of the lambda phages from fragment 01 to fragment 50 against these alternative restriction enzyme sites, and as a result, aarI was 12, bbsI was 24, bfuAI was 41, bsmFI was 38, btgZI was 45, and BsmBI was 14, and for any of the restriction enzymes, there was no restriction enzyme recognition site that was not present in the lambda phage genome. Therefore, restriction enzymes that do not cleave the inside are individually selected and used for each unit DNA. Combinations of restriction enzymes were investigated in order to reduce the types of restriction enzymes used as much as possible, and as a result, it was confirmed that only 3 types of BbsI, aarI, and BsmBI were used to satisfy the requirements. The type IIS restriction enzymes used for excising the unit DNAs were assigned as follows.

The BbsI cuts the groups of 01-08, 12, 16-22, 24, 27, 28, 33-39, 43, 45-50 fragments, and the total of 33 fragments; the group cut by AarI is the 09 th to 11 th, 13 th, 23 th, 25 th, 30 th, 32 th, 44 th fragments, the total of 9 fragments; the set cut with BsmBI was the 14 th, 15 th, 26 th, 29 th, 31 th, 40 th to 42 th fragments, totaling 8 fragments.

< cloning of Gene fragment >

All 50 fragments from fragment 01 to fragment 50 were amplified using the lambda phage genome full length using PCR method. First, a restriction enzyme recognition site determined as described above is added to the 5 '-end of a primer for amplifying a DNA sequence between the combinations of overhangs determined as described above so that the site is located at a position where the overhang is desired to be cut out, and further, a primer having a TAG sequence added to the 5' -end is used. Using these primer sets, DNA fragments of the designated region were amplified using the full length of the lambda phage genome. The reaction conditions for PCR were as follows: each 1 piece ofTo the reaction, 5. Mu.l of KOD Plus10 XBuffer Ver.2 and 3. Mu.l of 25mM MgSO 2 were added ₄ 5. Mu.l dNTPs (each 2 mM), 1. Mu.l KOD Plus (1 unit/. Mu.l), 48pg lambda phage DNA (TOYOBO), 15pmol primers (the concentrations of the F primer and the R primer), and sterile water were used to prepare a reaction System (50. Mu.l), which was then subjected to the following procedure using GeneAmp PCR System 9700 (Applied Biosystems).

After incubation at 94 ℃ for 2min, 30 cycles of 1 cycle of 10s at 98 ℃, 30s at 55 ℃ and 1min at 68 ℃ were performed, and then incubation at 68 ℃ for 7min was performed. In a 1% agar gel (Ultra Pure Agarose, invitrogen) prepared by diluting 50-fold with a 1 XTAE buffer solution (MilliQ water prepared by "Tris-acetic acid-EDTA buffer stock solution (50-fold concentrated) pH8.3 (at 25 ℃) manufactured by Nacalai Tesque Co., ltd.") containing 2mg/ml of crystal violet (Wako Pure chemical industries, ltd.), the amplified unit DNAs were separated over a running time of 10min with an electrophoresis apparatus (i-MyRun. NC, cosmobio), and bands of the target DNAs in the running gel were collected with a blade to obtain gel fragments of about 200 mg. Unit DNA was purified from the Gel fragment using the concentrate Rapid Gel Extraction System (Life Technologies Co., ltd.). Specifically, L1 Buffer was added to the gel pieces in a volume value of 3 times the weight of the gel, the gel pieces were dissolved in an incubator (block incubator) at 45 ℃ for about 10 minutes, the solution was added to a filter cartridge (spin column cartridge) attached thereto (a member having a purification column attached to a 2ml centrifuge tube), the solution was centrifuged at 20,000 × g for 1 minute and the liquid (flow-through) was discarded, then 750 μ L2Buffer was added to the purification column, and the liquid was centrifuged at 20,000 × g for 1 minute and the liquid was discarded. In order to remove the residue such as L2Buffer remaining in the column more reliably, the column was centrifuged again at 20,000 Xg for 1min, and the 2ml centrifuge tube used up to this stage was discarded, and the column was transferred to a new 1.5ml centrifuge tube. To the purification column, 30. Mu.l of TE buffer (10 mM Tris-HCl,1mM EDTA, pH 8.0) was added and left for 2min, followed by centrifugation at 20,000 Xg for 1min to recover the DNA solution. The recovered DNA was stored at-20 ℃ before use. The resulting unit DNA was cloned into an E.coli plasmid vector by the TA cloning method shown below.

Mu.l of 10 XEx-Taqbuffer attached to the enzyme Ex-Taq for PCR reaction of TAKARA, 0.5. Mu.l of 100mM dATP, and 0.5. Mu.l of lEx-Taq were added to 8. Mu.l of the unit DNA solution, and the mixture was incubated at 65 ℃ for 10min to attach a overhang A to the 3' -end of the unit DNA. Mu.l of the unit DNA solution was mixed with 1. Mu.l of pMD19-Simple from TAKARA and 3. Mu.l of sterilized water, and 5. Mu.l of TAKATA light (Mighty) Mix was added thereto, and the mixture was incubated at 16 ℃ for 30min. Mu.l of the ligation solution was added to 50. Mu.l of chemically competent cells of E.coli DH 5. Alpha. And incubated on ice for 15min, then heat shock (heat shock) was applied at 42 ℃ for 30sec, and after being left on ice for 2min, 200. Mu.l of LB medium was added and incubated at 37 ℃ for 1h, and then applied to LB plate medium (LB plate) containing carbenicillin (100. Mu.g/ml) and 1.5% agar and cultured overnight at 37 ℃ to obtain a transformant of the plasmid. The obtained colonies were prepared using a template DNA preparation reagent for PCR (Cica Geneus DNA preparation reagent, kanto chemical Co.). Specifically, 2.5. Mu.l of the resulting solution was prepared by mixing the reagent a and the reagent b in the kit at a ratio of 1: 10, and a small amount of colonies on a plate medium were collected with a toothpick, suspended in the solution, and then treated at 72 ℃ for 6min and 94 ℃ for 3min. To the resulting solution were added 2.5. Mu.l of TAKARA Ex-Taq 10 Xenzyme, 2. Mu.l of 2.5mM dNTP solution, 0.25. Mu.l of 10 pmol/. Mu.l M13F primer, 0.25. Mu.l of 10 pmol/. Mu.l M13R primer, 17. Mu.l of sterile water, and 0.5. Mu.l of Ex-TaqHS, and after incubation at 94 ℃ for 5min, DNA was amplified by performing 30 cycles at 98 ℃ for 20sec, 55 ℃ for 30sec, and at 72 ℃ for 1min, and the nucleotide sequence of the PCR product was examined to determine whether or not the PCR product completely coincided with the desired sequence. Finally, the correct sequence was obtained using all colonies. In this process, among the mutants obtained for fragment 10, there were synonymous substitution mutants (g.9515G > C) within the coding region of 1 gene V. This mutation led to the new emergence of the restriction enzyme AvaI recognition site in the phage genome (fig. 2). In this example, in order to clearly express that the constructed phage was a phage created artificially, the synonymous substitution mutant (g.9515G > C) was used for fragment 10 without using the wild type.

< purification of plasmid having Unit DNA in high purity >

Coli transformants containing the plasmids into which the 01. Sup. Th to 50. Sup. Th fragments having the desired sequences were cloned were cultured overnight at 37 ℃ and 120spm in 50ml of LB medium supplemented with 100. Mu.g/ml carbenicillin, and the obtained cells were purified using QIAfilter Plasmid Midi Kit (QIAGEN). To 50. Mu.l of the crude plasmid solution obtained, 5. Mu.l of 3M potassium acetate-acetic acid buffer (pH 5.2) and 125. Mu.l of ethanol were added, the mixture was centrifuged at 20000 Xg for 10min, DNA was precipitated with ethanol, the resulting precipitate was washed with 70% ethanol, and the residue was removed and dissolved in 50. Mu.l of TE (pH 8.0) again. To measure the concentration, 1. Mu.l of the crude extract plasmid solution was taken and the DNA concentration was measured by an ultramicro absorptiometer (ND-2000, thermo Co.). At this time point, the amount of DNA in the crude extract plasmid solution was approximately 0.5 to 4. Mu.g/. Mu.l. Referring to the measured values, 5. Mu.g of DNA was collected from each crude extracted plasmid solution into a 1.5ml tube, and sterilized water was added so that the total volume of each solution became 50. Mu.l. To the solution, 6. Mu.l of a 10 × reaction buffer solution of Plasmid Safe (Epicentre Co.), 2.4. Mu.l of a 25mM EDTA solution and 2. Mu.l of a Plasmid Safe enzyme solution were added and mixed, and then the mixture was incubated at 37 ℃ for 1 hour by using a programmable incubator (programmable block incubator) BI-526T (ASTEC Co.), followed by incubation at 75 ℃ for 30 minutes for inactivating the enzyme. The resulting solution was purified using a PCR purification kit (QIAGEN). In the final purification stage of the kit, the DNA adsorbed to the purification column was eluted with 25. Mu.l of TE buffer (pH 8.0) without using the elution buffer attached to the kit, to obtain a high-purity plasmid solution. DNA electrophoresis (Ultra Pure Agarose, invitrogen) was performed using the plasmid having the 01. Sup. Th fragment and the plasmid having the 21. Sup. St fragment before and after purification, and it was confirmed that the target fragment (unitary DNA) had been integrated (FIG. 3).

< precise concentration adjustment and equimolar integration of plasmid having Unit DNA >

The resulting DNA solution was measured again by an ultramicro absorptiometer to determine the concentration of the high purity plasmid solution. The concentration of each sample reflects the degree of purification of the crude extracted plasmid solution, compared to the theoretical maximum, i.e., 200 ng/. Mu.l, and ranges from about 100 ng/. Mu.l to 200 ng/. Mu.l. Based on the measurement results, 15. Mu.l of each plasmid solution was taken out into a 1.5ml tube, TE was added to each solution so that the concentration of each plasmid became 100 ng/. Mu.l, and the obtained high purity plasmid solution was measured again with an ultramicro absorptiometry, and as a result, since there was an error in a range of about several percent with respect to 100 ng/. Mu.l as a target value, a volume amount of 500ng of DNA was calculated with an accuracy of μ l at 2 decimal places, each DNA solution was divided into the volume amounts (about 5. Mu.l), and the respective DNA units were integrated according to the types of restriction enzymes (BbsI group, aarI group, bsmBI group) to be used for excision thereafter, so that the number of moles of each unit DNA was substantially equalized.

< collective cleavage of an equimolar integration plasmid Using restriction Endonuclease >

Regarding the total volume of the integrated equimolar plasmid solution, the BbsI group was about 165. Mu.l, the AarI group was about 45. Mu.l, and the BsmBI group was about 40. Mu.l. To each group, 2-fold sterilized water was added to obtain 495, 135 and 120. Mu.l of high purity plasmid solutions, respectively, and the plasmid solutions were cleaved as follows according to the type of restriction enzyme.

For the BbsI group, 55. Mu.l of 10 XNEBbuffer #2 and 27.5. Mu.l of restriction enzyme BbsI (NEB Co.) were added, totaling about 577. Mu.l, and reacted at 37 ℃ for 2 hours. For the AarI group, 15. Mu.l of 10 XBuffer _ for _ AarI attached to the restriction enzyme, 3. Mu.l of 50 XOligonletide for activating cleavage also attached to the restriction enzyme, and 7.5. Mu.l of restriction enzyme AarI (Thermo Co.) were added thereto in a total amount of about 160. Mu.l, and reacted at 37 ℃ for 2 hours. For the BsmBI group, 13.3. Mu.l of 10 XNEBBuffer #3 and 6.3. Mu.l of restriction enzyme BsmBI (NEB Co.) were added, amounting to about 140. Mu.l, and reacted at 55 ℃ for 2h. After 2 hours, the plasmid solutions were collected from the respective samples in such a manner that the equimolar relationship was not impaired, namely, 33. Mu.l from BbsI group, 9. Mu.l from AarI group, and 8. Mu.l from BsmBI group, and by subjecting 5. Mu.l thereof to DNA electrophoresis, it was confirmed that the plasmids were cleaved with the respective restriction enzymes (FIG. 4).

< collective isolation and purification of 50 Unit DNAs by agarose gel electrophoresis >

After confirmation, phenol, chloroform and isoamyl alcohol (25: 24: 1) (Nacalai Tesque Co.) were added in equal amounts to each group, and the restriction enzymes were inactivated by thorough mixing. Here, after the phenol/chloroform/isoamyl alcohol (25: 24: 1) mixture of each group was integrated into 1 tube, the mixture was separated into a phenol phase and an aqueous phase by centrifugation (20,000 Xg, 10 min), and the aqueous phase (about 900. Mu.l) was recovered into another 1.5ml tube. To the aqueous phase was added 500. Mu.l of 1-butanol (Wako pure chemical industries, ltd.), well mixed, separated by centrifugation (20,000 Xg, 1 min), and the operation of removing the water-saturated 1-butanol was repeated to reduce the volume of the aqueous phase until the volume of the aqueous phase became 450. Mu.l or less. To this, 50. Mu.l of 3M potassium acetate-acetic acid buffer (pH 5.2) and 900. Mu.l of ethanol were added, DNA was precipitated by centrifugation (20,000 Xg, 10 min), and the precipitate was washed with 70% ethanol and dissolved in 20. Mu.l of TE. To this was added 2. Mu.l of 10 XDye for electrophoresis, and in the presence of 1 XTAE (Tris-Acetate-EDTA Buffer) Buffer, using 0.7% low melting point agar gel (2-Hydroxyethyl Agarose type VII, sigma), a voltage of 35V (about 2V/cm) was applied using a general agar gel electrophoresis apparatus (electrophoresis system for i-MyRun. N nucleic acids, cosmobio Co.), and the 01 th to 50 th fragments and the plasmid vector in the whole sample were separated by 4h electrophoresis (FIG. 5). The electrophoresis gel was stained with 100ml of 1 XTAE buffer containing 1. Mu.g/ml ethidium bromide (Sigma Co.) for 30min, visualized by irradiation with ultraviolet light of long wavelength (366 mn), and the band formed by the 01. Sup. Th to 50. Sup. Th fragments (about 1kb close) was cut out with a razor blade and collected in a 1.5ml tube. To the recovered low melting point agar gel (about 300 mg), 1 XTAE buffer was added to make the total volume about 700. Mu.l, and the gel was dissolved by keeping it at 65 ℃ for 10 min. To the obtained gel solution, 500. Mu.l of 1-butanol was added, the aqueous phase and butanol phase were separated by centrifugation (20,000 Xg, 1 min), and water-saturated butanol was repeatedly discarded until the volume of the aqueous phase became 450. Mu.l or less. To the resulting solution were added 50. Mu.l of 3M potassium acetate-acetic acid buffer (pH 5.2) and 900. Mu.l of ethanol, and DNA precipitates were obtained by centrifugation (20,000 Xg, 1 min), and the precipitates were washed with 70% ethanol and dissolved in 20. Mu.l of TE. Mu.l of the resulting solution was taken and the concentration was measured by an ultramicro absorptiometer.

In order to confirm the number of moles of each group before and after size fractionation, quantitative PCR was performed. FIG. 6 shows the distribution of the number of molecules of each unit DNA before and after size fractionation, and FIG. 7 shows the rate of change in the number of molecules of each unit DNA. Thus, it was confirmed that 50 fragments were recovered in a substantially equimolar ratio without impairing the molar ratio.

< Gene Assembly >

The equimolar mixture of the 01 st to 50 th fragments had a DNA weight concentration of 98 ng/. Mu.l, a total base sequence of 48 and 522bp, and the vector DNA (pGETS 118-AarI/AarI) of 190 ng/. Mu.l and a total length of 15 and 139bp. Based on the weight ratio of the length, the vector DNA was mixed in a ratio of 1.00. Mu.l to 6.21. Mu.l of the equimolar mixture of the 01. Mu.l to 50. Mu.l of the two types of DNA. Mu.l of 2 Xligation buffer was added to 7.2. Mu.l of the resulting equimolar mixture, and the whole mixture was incubated at 37 ℃ for 5 minutes, followed by addition of 1. Mu.l of T4DNA ligase (Takara) and incubation at 37 ℃ for 4 hours. A part of the gel was electrophoresed to confirm that the gel was connected (FIG. 8). Mu.l of the suspension was collected in a new tube, and 100. Mu.l of Bacillus subtilis competent cells were added thereto, followed by rotary culture at 37 ℃ for 30min using a rotary mixer (duck rotor). Then, 300. Mu.l of LB medium was added, and the mixture was subjected to rotary culture at 37 ℃ for 1 hour using a rotary mixer, and then the culture solution was applied to LB plate medium containing 10. Mu.g/ml tetracycline, and the culture was carried out overnight at 37 ℃. 250 colonies were obtained.

< confirmation of plasmid Structure of transformant >

12 colonies were randomly selected, cultured overnight in 2ml of LB medium containing 10. Mu.g/ml of tetracycline, IPTG was added at a final concentration of 1mM in order to increase the copy number of the internal plasmid, and further cultured at 37 ℃ for 3 hours. Plasmids were extracted from the obtained cells, digested simultaneously with restriction enzymes HindIII and SfiI, and electrophoresed to confirm that 4 of 12 strains showed the desired cleavage pattern (FIG. 9). For these 4 strains, plasmids were prepared in large quantities by cesium chloride ethidium bromide density gradient ultracentrifugation, treated with 13 restriction enzymes, and the structures of the plasmids were confirmed by electrophoresis, and were consistent with the expected fragments (FIG. 10). Further, the nucleotide sequence was determined for all regions of the plasmid except for the vector portion, and as a result, the nucleotide sequences of all 4 plasmids were completely identical to the expected nucleotide sequences.

< confirmation of function of assembled Gene >

To confirm the function of the 4-strain plasmid as lambda phage, plaque-forming ability was confirmed as described below. First, each of the assembled plasmids of #3, #4, #6 and #12 was cleaved with Lambda terminator (Lambda terminator, epicenter) to divide the plasmid into a vector and an assembled gene portion, which was added to Lambda Packaging Extract (Gigapack III Plus Packaging Extract, agilent Technologies). Escherichia coli (VCS 257 strain) was infected, spread on LB plate medium, and cultured overnight at 37 ℃ to confirm plaque. The shape of the plaque obtained was confirmed to be the same as that of a plaque obtained by using phage lambda DNA manufactured by TOYOBO Co., ltd, which was carried out in parallel (FIG. 11). Phage DNAs were purified from plaques obtained using each plasmid, and the presence or absence of the introduced mutation was confirmed by cleavage with the restriction enzyme AvaI, and as a result, as shown in FIG. 12, the cleavage pattern was different from that of the lambda phage DNA manufactured by TOYOBO, and it was confirmed that AvaI site was present in all phages as expected. From this, it was confirmed that the nucleotide sequence and plaque-forming ability of the lambda phage genome prepared by assembling 50 fragments in total of the 01. Sup. Th to 50. Sup. Th fragments were all intact.

The above results indicate that 50 unit DNAs constituting the lambda phage DNA and 51 DNA fragments in total of the vector DNA (pGETS 118-AarI/AarI) can be linked.

Example 2 construction of mevalonate pathway Artificial operon Using an Assembly of 55 Unit DNAs and vector DNAs

As a substance having an isoprene monomer as a skeleton, such as isoprenoid, various substances are known, and these substances are each synthesized from a common material, isopentenyl diphosphate (IPP). It is known that there are 2 pathways from glycolysis to IPP, namely, the mevalonate pathway and the non-mevalonate pathway, and that only the non-mevalonate pathway is present in escherichia coli, although there are also organisms having both pathways in a single organism. In order to enhance the IPP-producing ability of E.coli, it was attempted to construct an artificial gene adapted to the codon usage frequency of E.coli for a part of the gene of mevalonate pathway of a yeast, a eukaryote, by assembling using a synthetic DNA fragment.

< sequence design of the Artificial mevalonate operon >

An attempt was made to transform codons of 3 genes (ERG 10 (1.2 kb), ERG13 (1.5 kb), and HMG1 (3.2 kb)) required for the metabolic pathway from acetyl CoA to mevalonate in the first half of the mevalonate pathway in yeast based on the codon usage frequency in E.coli, and a manual operator (5,951bp) (SEQ ID NO: 4) was prepared by aligning 3 manual genes (the entire length of SEQ ID NO: 4 was 5,955pb which is a sequence including 4 bases for protrusion). The yeast gene was transformed into E.coli codons by the following procedure: in yeast synonymous codons, the codons were ranked according to the frequency of occurrence of the codons in all the genes in yeast, and similarly, in Escherichia coli synonymous codons, the codons having the same rank were ranked according to the frequency of occurrence in all the genes in Escherichia coli, and the codons were exchanged.

< design of Unit DNA >

As a result of searching for a restriction enzyme site that does not cleave a 5,951bp DNA sequence transformed with synonymous codon in the same manner as in example 1, it was found that no recognition sequence of the restriction enzyme AarI is present and no cleavage by AarI occurs, and thus all clones were prepared using AarI. Since fragments having an average size of 108bp were obtained by dividing 5,951bp in total into 55 fragments, it was examined whether or not a specific sequence (44 combinations (group III) obtained by excluding 8 palindromic combinations from 52 combinations of 2 in total of a and T and 2 in total of C and G), and 16 combinations (group IV) obtained by excluding three consecutive C and G from 32 combinations of 3 in total of a and T and 3 in total of C and G, and 60 in total of 2) appeared in the vicinity of the ideal division unit, using this size as an ideal division unit, and it was found that any one of specific sequences appeared within a range of ± 7bp from the ideal division unit. Based on the results, the total length was divided into 55 fragments of 98 to 115 bp. Table 2 shows the DNA assembled in example 2 in terms of the divided units and the protruding base sequences. Note that for the demarcation of mevalonate gene groups and gene assembly vectors, the protrusions consisting of only a and T (ATTA and AAAA) were used.

[ TABLE 2 ]

< preparation of Unit DNA Using synthetic DNA >

Each of the fragments obtained by segmentation was prepared using 2 segments of 80-base chemically synthesized DNA by the method of Rossi et al (Rossi, J.J., and Itakura, K.1982.J.biol.chem.257, 9226-9229 (1982)). Specifically, 2 pieces of chemically synthesized DNA were hybridized with several tens of bp at the 3' -end, and a recognition site was added to the 5' -end, so that the overhang designed as described above appeared on the 5' -end side of the AarI cleavage site by AarI digestion. These 2 pieces of synthetic DNA hybrid products and 1 kind of subsequent template based extension reaction obtained double-stranded unit DNA by PCR amplification to prepare with two terminal AarI recognition sites hybridization PCR primers (total 3 kinds of DNA), through PCR reaction to obtain two ends with AarI cutting sites unit DNA, using TA cloning method and Escherichia coli plasmid vector pMD19 connected, through transformation and cloning to Escherichia coli. By sequencing the ligated plasmid, a clone having a desired nucleotide sequence was selected for each fragment.

< equimolar mixing of plasmids having Unit DNAs >

55 E.coli containing the desired clone thus obtained were cultured, and 50. Mu.l of a crude extracted Plasmid solution was obtained from each strain by using Plasmid mini-prep (QIAGEN). Mu.l of each solution was taken and the DNA concentration was measured by an ultramicro absorptiometer to find that the concentration was 82 to 180 ng/. Mu.l. About 5. Mu.g of each Plasmid was treated with Plasmid Safe, and after heat inactivation of the enzyme, the Plasmid was purified with Mini-enzyme PCR purification Kit (QIAGEN Co.) to obtain 25. Mu.l of a high purity Plasmid solution. The concentration of 1. Mu.l of the solution was measured by an ultramicro absorptiometer, and the concentration was 108 to 213 ng/. Mu.l. From each solution, 20. Mu.l of the high purity plasmid solution was taken out to other tubes, and TE was added to these tubes for dilution so that the concentration of each plasmid was calculated to be 100 ng/. Mu.l. The concentration of the purified plasmid solution was calculated again by microabsorption photometry, and the volume of each high-purity plasmid solution was calculated at a weight of 500ng with a precision of. Mu.l at 2 decimal places based on the concentration, and the volume (about 5. Mu.l) was taken out from each plasmid solution and collected in one tube. To about 275. Mu.l of the plasmid mixture solution in total, 2 times the volume of sterile water, 137.5. Mu.l of 10 XBuffer for AarI, and 67.5. Mu.l of restriction enzyme AarI were added and reacted at 37 ℃ overnight.

size-Co fractionation of < 55 Unit DNAs >

After inactivating AarI by adding an equal amount of phenol, chloroform and isoamyl alcohol (25: 24: 1) to the reaction solution, centrifugation was performed, the supernatant was purified by ethanol precipitation, and the precipitate was dissolved in 20. Mu.l of TE. Xylene nitrile blue as a pigment for electrophoresis was added to the solution, and electrophoresis was performed on a 2.5% agar gel at 100V for 30 minutes using TAE as a buffer solution to separate pMD19, which is a vector DNA, from unit DNA, which is an insert gene (FIG. 13). The electrophoresed gel was cut with a razor blade, a part of the gel was stained with ethidium bromide, and a band of the target DNA was excised from the unstained gel while confirming the position of the band of the 55 equimolar mixture fragments as the target.

< purification of equimolar Unit DNA population >

The DNA was purified from the Gel fragment obtained using the MiniElute Gel Extraction Kit (QIAGEN Co.) as described below.

After the volume of the gel was determined by measuring the weight thereof, CG Buffer was added in an amount 15 times the volume of the gel, and the temperature was maintained at 50 ℃ for 10min to dissolve the gel. To the dissolved gel, isopropanol was added in an amount 5 times the volume of the gel, and the resulting solution was introduced into an attached purification column, and the DNA was adsorbed to the purification column by centrifugation. To the purification column was added 500. Mu.l of CG Buffer, and after washing by centrifugation, 750. Mu.l of PE Buffer was further added, and washing by centrifugation was carried out. To completely remove the residue, centrifugation was performed 1 time, 10. Mu.l of TE buffer was added to the column, and the mixture was centrifuged to obtain a 55-fragment unit DNA mixture solution having approximately the same molar number.

< addition of DNA having an origin of replication to an equimolar Unit DNA mixture solution >

The DNA concentration was measured by an ultramicro absorptiometer and found to be 20 ng/. Mu.l. pGET151/AarI prepared in parallel was at a concentration of 67 ng/. Mu.l, and therefore, an equimolar mixed solution of the 55 fragment and pGETS151/AarI was mixed so that the ratio of the two was 4.63: 1, taking into account the ratio of the lengths of the two (5955bp.

< Gene Assembly >

Mu.l of 2 Xligation buffer was added to 5.63. Mu.l of the resulting equimolar mixture, and the whole was incubated at 37 ℃ for 5 minutes, followed by addition of 1. Mu.l of T4DNA ligase (Takara) and incubation at 37 ℃ for 4 hours. A part of the DNA fragments was subjected to electrophoresis to confirm whether the unit DNA and the vector DNA were ligated in tandem (FIG. 14). Mu.l of the ligated solution was collected into another tube, and 100. Mu.l of Bacillus subtilis competent cells were added, followed by rotary culture at 37 ℃ for 30min using a rotary mixer. Then, 300. Mu.l of LB medium was added, and after 1 hour of rotary culture at 37 ℃ using a rotary mixer, the culture was applied to LB plate medium containing 10. Mu.g/ml tetracycline.

< confirmation of the structures of the transformants and the assemblies >

From the resulting 154 colonies, 24 clones were randomly selected and inoculated in LB containing 10. Mu.g/ml of tetracycline. After IPTG was added to a final concentration of 1mM in the logarithmic growth phase and cultured to a stable phase, plasmid DNA was extracted and treated with restriction enzyme PvuII, and the cleavage pattern was investigated by electrophoresis (fig. 15). As a result, it was confirmed that 2 clones (# 10 and # 20) matched the predicted nucleotide sequence, and therefore, these plasmids were subjected to other restriction enzyme treatments, and the more detailed structure was confirmed by electrophoresis, and as a result, the plasmids matched the target structure (FIG. 16). This plasmid was sequenced, and it was finally confirmed that clones 10 and 20 had a base sequence consistent with the design.

The above results indicate that a total of 56 DNA fragments consisting of 55 unit DNAs constituting the mevalonate pathway artificial operon and vector DNA (pGETS 151-pBR) can be ligated.

From the above results, it was confirmed that 50 or more DNA fragments can be ligated by the method for preparing a DNA conjugate of the present invention. It is considered that the reason why a plurality of DNA fragments can be ligated in this way is that the number of moles of each unit DNA is more accurately approximated to the same in the unit DNA composition prepared by the preparation method according to the present invention.

The reason why the number of moles of each unit DNA in the unit DNA composition prepared by the preparation method according to the method of the present invention is more accurately close to the same is inferred to be as follows.

In examples 1 and 2, when the concentration of each unit DNA in a solution containing the unit DNA is measured, an additional sequence (specifically, circular plasmid DNA) is ligated to each unit DNA. Therefore, even if the distribution of the lengths of the respective base sequences among the unit DNAs is large, the distribution of the lengths of the base sequences decreases by a certain amount when the concentration of the solution is measured because the additional sequences are linked. Therefore, the error in the number of moles of each unit DNA calculated based on the measurement result is reduced. Therefore, by dividing each solution based on the measurement result and adjusting the number of moles of unit DNA in each solution to be the same, the molar ratio in each solution is easily approximated to 1, and the number of moles of unit DNA is more accurately approximated to be the same.

In example 1, the standard deviation of the distribution of the total length of each base length of the unit DNA and the total base length of the additional sequence ligated to each unit DNA was 3691.4. + -. 6.6bp, and. + -. 0.18% of the average total length. In example 2, the standard deviation of the distribution of the total length of each base length of the unit DNA and the total base length of the additional sequence ligated to each unit DNA was 2828.2. + -. 4.5bp, and. + -. 0.16% of the average total length. In examples 1 and 2, as described above, the ratio of the standard deviation to the average total length is small, and therefore, the error in the number of moles of each unit DNA calculated based on the measurement result of the concentration of DNA in the solution is considered to be small.

The ratio of the average base length of the additional sequence linked to each unit DNA to the average base length of the unit DNA was about 2.7 in example 1 and about 27 in example 2. In this way, it is considered that, since the average base length of the additional sequence linked to each unit DNA is longer than the average base length of the unit DNA, the error in the number of moles of each unit DNA calculated based on the measurement result of the concentration of DNA in a solution is smaller, and the error in the number of moles of each unit DNA calculated based on the measurement result of the concentration of DNA in a solution is smaller.

It is also shown in examples 1 and 2 that since the DNA is designed with a non-palindromic sequence located in the vicinity of the sequence at the position where the assembled DNA is divided into equal parts (the assembled DNA is divided into equal parts such that the base lengths of the parts are equal when the base length of the sequence of the assembled DNA is divided by the number of types of the unit DNA), the unit DNA is designed such that the lengths of the unit DNAs are substantially the same. Therefore, in the size fractionation after electrophoresis after removal of the additional sequence by the restriction enzyme, bands at approximately the same position appear, and the unit DNA can be recovered by 1 size fractionation, thereby improving the operation efficiency.

In example 1, restriction enzymes used for removal of additional sequences were classified into groups (3 in example 1 and 1 in example 2). Before the removal step, 2 or more types of unit DNA-containing solutions for each set may be mixed, and the restriction enzyme treatment may be performed once for each restriction enzyme set without performing the restriction enzyme treatment separately for each unit DNA. Thus, it was confirmed that the operation efficiency of the preparation of DNA links was improved. It was also confirmed that even when the solutions thus mixed were used, as described above, since the solutions were mixed in a state in which the number of moles was substantially equal, a large number of unit DNAs could be linked.

Test example 1 confirmation of redundancy of the repetitive units (redundancy (r)) of the number of assembling units DNA required for transformation of Bacillus subtilis plasmid DNA

In order to confirm the number of repeats (redundancy) r of the assembly unit DNA required for transformation of Bacillus subtilis plasmid, the following experiment was performed.

Using plasmid pGETS118-t0-Pr-SfiI-pBR (SEQ ID NO: 1) having a replication origin effective in Bacillus subtilis, the following DNAs (A) to (H) were prepared.

< preparation of DNA of (A) >

(A) The DNA of (3) is a circular monomeric plasmid DNA with redundancy r =1. First, pGETS118-t0-Pr-SfiI-pBR was transformed into E.coli. The plasmid obtained from this transformant is mainly the DNA of (a), but contains a certain amount of multimers (multimers), and therefore, in order to remove the multimers, the plasmid was subjected to DNA size fractionation by low melting point agar gel electrophoresis, and only the gel of the region of the monomer plasmid DNA was excised and purified, thereby preparing the DNA of (a).

Production of DNA of (B)

(B) The DNA of (3) is a linear monomeric plasmid DNA with redundancy r =1. The DNA of (B) was prepared by treating the DNA of (A) above with the restriction enzyme BlpI (recognition site of (5 '-GC/TNAGC-3')).

< preparation of DNA of (C) >

(C) The DNA of (1) is a linear multimeric plasmid DNA of tandem repeats having a redundancy r > 1. BlpI used for preparing the DNA of the above (B) forms a non-palindromic 3-base overhang at the 5' -end. Thus, by ligating the DNAs of (B) above with a DNA ligase, a linear multimeric plasmid DNA in which plasmid units are continuous in the same direction, that is, the DNA of (C), is prepared.

< preparation of DNA of (D) >

(D) The DNA of (3) is a linear monomeric plasmid DNA with redundancy r =1. The DNA of (D) was prepared by treating the DNA of (A) above with the restriction enzyme EcoRI (recognition site: 5 '-G/AATTC-3')).

< preparation of DNA of (E) >

(E) The DNA of (4) is a linear multimeric plasmid DNA which locally contains a redundant r > 1 portion and is formed by linking the DNAs of (D) in random orientations. EcoRI used for preparing the DNA of the above (D) forms a palindromic 3-base overhang at the 5' -end. When plasmid DNAs cleaved with EcoRI are ligated, multimeric plasmid DNAs in which plasmid units are ligated in random orientations can be prepared. (E) The DNA of (4) is prepared by ligating the DNAs of (D) above with a DNA ligase.

< preparation of DNA of (F) >

(F) The DNA of (a) is prepared by the following method

The linear quasimonoid mixture of (a): a DNA fragment obtained by cleaving only 1 part of the DNA of (A) with the cleavage restriction enzyme KasI and cleaving with BlpI in the vicinity thereof after dephosphorylation, and a DNA fragment obtained by cleaving only 1 part of the DNA of (A) with the restriction enzyme AfeI and cleaving with BlpI in the vicinity thereof after dephosphorylation were mixed in equal amounts. The redundancy r of any of the DNA fragments in the mixture of (F) is slightly lower than 1.

Production of DNA of < G >

(G) The DNA of (4) is a linear quasi-dimeric plasmid DNA with redundancy r =1.98, which is obtained by ligating the 2 DNA fragments of (F) above using a DNA ligase and ligating them with a direction designated only by the BlpI site.

< preparation of DNA of (H) >

The cleavage sites of the above (B) and (D) are separated from each other. (H) The DNA of (4) is a mixture of the DNAs of (B) and (D) in such a manner that the number of moles of the DNAs is equal to each other and the DNAs are mixed in a non-linked state.

Transformation of Bacillus subtilis competent cells with DNAs of (A) to (H) >

The DNAs of the above (A) to (H) were transformed into competent cells of Bacillus subtilis, and transformants were obtained for each 1. Mu.g using the number of occurrences of the obtained tetracycline-resistant strain as an index. The DNAs of (A) to (H) were dissolved in a ligation buffer and used for transformation, regardless of whether the ligation reaction was performed. (A) FIGS. 17 are photographs showing the electrophoresis of DNAs (A) to (H) showing the number of transformants of Bacillus subtilis competent cells of the DNAs (A) to (H) shown in FIG. 18. In the electrophotograph of FIG. 17, the DNA of (C) and (E) has fragments of various sizes distributed over a wide range on the lane, and therefore the bands are not easily distinguished. In fig. 17, "G", the upper band is DNA (G) with redundancy r =1.97, and the lower band is DNA (G) with redundancy r =0.95 mixed with DNA (G).

From the above results, it was confirmed that only the DNAs (C), (E) and (G) obtained by ligating the DNAs were obtained from the transformant except for the circular DNA of A. This indicates that, when the redundancy r =1 or r < 1, even when 2 kinds of linear plasmid molecules having different cleavage sites and a relationship in which sequences capable of complementing the cleavage sites are present are mixed, a transformant cannot be obtained, and that, at least in linear DNA, the minimum redundancy must satisfy r > 1.

(simulation 1 connection simulation)

< setting of connection simulation Algorithm >

Simulation programming was performed using VBA of table computing software Excel (registered trademark) 2007. Using 3 parameters F _i (N _i ，L _i ，R _i ) The expression mimics the ligated DNA fragment F. Here, "i" represents an identification number of a fragment, more specifically, an i-column cell on Excel. "N" represents the number of unit DNA fragments contained in 1-molecule ligated DNA fragment in the mock ligation, "L" represents the result of digitizing the sequence of the left protruding end of the ligation product by an arbitrary natural number, and "R" represents the result of digitizing the sequence of the right protruding end of the ligation product by an arbitrary natural number in the same manner as "L". Here, when L = R, 2 overhanging sequences are in a complementary relationship, and L = R is defined as being capable of ligation. The simulation of the connection is performed as follows.

For F _i (N _i ，L _i ，R _i ) A segment, which is a uniform random number (uniform random number) between 0 and 1 generated by a RAND () method (method) multiplied by m (described later), is rounded to generate a random number j satisfying i ≠ j, and F is selected based on the number _j (N _j ，L _j ，R _j ) And (3) fragment. Whether or not these 2 segments can be connected is determined using the following formula, and when connection is possible, the parameters of the segments are changed as follows.

Satisfy L _i ＝R _j When the left end of Fi and the right end of Fj can be connected, it is converted to F _i(new) (N _i(old) +N _j(old) ，L _j(old) ，R _i(old) ) Fragment and F _j(new) (0,0,0) fragment. Otherwise, satisfy R _i ＝L _j When is at F _i Right end of fragment and F _j When the left ends of the fragments can be ligated, they are converted to F _i(new) (N _i(old) +N _j(old) ，L _i(old) ，R _j(old) ) Fragment and F _j(new) (0,0,0) fragment. L is _i ≠R _j And R is _i ≠L _j Is unconverted (F) _i(new )(N _i(old) ，L _i(old) ，R _i(old) ) Fragment and F _j(new) (N _j(old )，L _j(old) ，R _j(old) ) Fragment), no analog ligation occurred. For the analog connection, the above calculation until i =1 → m is performed as 1 cycle. Here, m is a variable representing the total number of DNA fragments in the mock ligation cycle, and also represents the initial total number of unit DNA fragments at the 1 st cycle of the mock. After 1 simulation connection cycle was calculated, F was changed using the Sort function (Sort Method) of VBA command of Excel2007 _i The fragments are arranged such that L _i The values are in descending order, counting F (0,0,0) outside of the F fragment _i The total number of fragments, embedded as new m, were subjected to the next round of analog ligation. The simulation connection is circulated until m is the minimum number of fragments that have no more outstanding fragments with complementary relationship and can not continue to perform simulation connection _min Until now. Here, m _min The value was obtained by the following calculation using the information of the initial unit DNA fragment before the start of ligation.

m _min = (total number of unit DNA fragments) one (total number of unit DNA fragments in the entire system with less number of unit DNA fragments among 2 kinds of unit DNA fragments satisfying the relationship of L = R)

< connection simulation >

In each assembly scale from assembly of 6 fragments, 13 fragments, 26 fragments, and 51 fragments, a group of dummy unit DNA fragments having a Coefficient of Variation (CV) set in the range of 0 to 20% with 1% as a scale was prepared as follows, and the average number of the same unit DNA fragments was 640.

Using a uniform random number command RAND () of Excel, a random number group of 0 to 1 is generated in an amount corresponding to each assembly scale, the random number group is normalized,the mean was made 0 and the variance was made 1, and then each normalized random number was multiplied by (the segment mean) ^* CV (%)/100), and the obtained value was added to the average value of the number of fragments, thereby preparing each analog unit DNA fragment group. For the random number groups, 20 independent groups were created for each CV (%) value of each assembly scale, and the above simulation was performed for each random number group, and the simulation was connected to m _min Until now. The 20 obtained simulated ligated fragments were integrated, and the number of ligated fragments was integrated for each value of N to determine the ratio of (N value. Times. The number of ligated fragments) to the total number of unit DNA fragments used for ligation, thereby creating a 100% stacking diagram so that the value of the molecule with the larger value of N is located at the lower side. This graph is shown in fig. 19. FIG. 19 shows the distribution of the sizes of the ligation products finally integrated with the original unitary DNA fragments. In FIG. 19, (a) is a graph showing 6 fragment assembly, (b) is a graph showing 13 fragment assembly, (c) is a graph showing 26 fragment assembly, and (d) is a graph showing 51 fragment assembly. In the case of 6-segment assembly, the regions where n =6, redundancy r =1, and redundancy r < 1 are shown in the upper right region. This result shows that, in the 6-fragment assembly, almost all unit DNA fragments were integrated into DNA fragments having a redundancy r > 1 region in the case of any CV value, whereas, in the 51-fragment assembly, almost all regions were regions having a redundancy less than 1 except for regions having CV close to 0%.

< derivation of connection theoretical equation from connection simulation >

When a general expression of the connection mechanism was obtained from the numerical analysis of the connection simulation described above, it was verified whether or not a fitting curve of the distribution of the connection product for each CV value at each assembly scale could be calculated. Fig. 20 shows the distribution of the numbers of DNA unit fragments contained in the ligation product at CV =20% in the case of 6-fragment assembly and 640 fragments on average. In fig. 20, the respective patterns of the columns of the respective redundancies (0 < r < 1, 1 < r < 2, 2 < r < 5, 5 < r < 10) indicate the types of the components classified by dividing N of the respective redundancies by r (5 components out of 6 components having remainders of 0, 1, 2, 3, 4, 5 when 6 segments are assembled, the components having remainders of 0, 1, 2, 3, 4, 5 which cannot be converted into logarithms because the value of N is 0 are removed), and the columns of the same patterns indicate the components classified by dividing N by r are of the same type in the different redundancies. The linear approximation curve in the graph of fig. 20 is obtained for each type of pattern component.

The histogram of the number of molecules of the ligation product of each N value shows a tendency of exponential decrease in the number of molecules with increase in N value as a whole, and it is found by preliminary observation that the geometrical distribution is approximated as one of discrete probability distributions. However, the histogram microscopically shows a periodic structure in which the number of scales or 1 redundancy (6 in the case of 6-segment assembly) of gene assembly is periodic, and particularly shows a characteristic structure in which no segment at all occurs in a portion where the value of N corresponds to an integral multiple of the scale of gene assembly. This feature does not coincide exactly with the geometric distribution or the exponential distribution when considered as a continuous probability distribution. However, since the axis indicating the number of molecules of the ligation product is converted to a logarithmic scale, and each component of the microscopic periodic structure (i.e., a component classified by the remainder obtained by dividing N by the assembly scale, i.e., 6) is extracted from each period and a linear approximation curve is obtained, the square value (0.94 or more) of the extremely high correlation coefficient is shown, and therefore, it is considered that each component has no problem even if it approaches the exponential distribution. This distribution was confirmed in the examples of other assembly scales and other CV values, in addition to the 6-segment assembly shown in fig. 20 in which the concentration difference CV was 20%. Then, assuming that the local system is heuristically close to the exponential distribution, the operation is performed for the exponential distribution function (f (n) = λ ^* exp(-λ ^* n)) of the image.

Specifically, (1) first, in all the simulations of the respective assembly scales described above, for each component classified by the remainder obtained by dividing N by the assembly scale, a straight line is obtained by linear approximation using a logarithmically converted numerator value of about 3 cycles, the slope of the straight line is obtained as- λ, and λ is calculated from the slope. (2) Next, in the exponential distribution function, 1/λ, which is the reciprocal of the parameter λ, is an average value of f (N), and therefore, the average value of the N values of the connected products in the 20 random number groups is obtained, and λ (CV (%)) of each CV (%) is calculated from the reciprocal of the average value. The results of (1) and (2) are plotted with respect to the CV (%) value of the concentration difference of the unit DNA fragments on the horizontal axis and the lambda value on the vertical axislot), it was confirmed that all plots exist on a direct proportional straight line passing through a certain origin regardless of the scale of gene assembly. From these plotted points, linear approximation curves are obtained, and the graph is shown in fig. 21. In FIG. 1, (a) is a graph showing the relationship between the slope λ obtained from the 3 cycles of (1) and the difference in concentration between the DNA unit fragments, and (b) is a graph showing the relationship between λ obtained from the reciprocal of the average value of the N values of (2) and the difference in concentration between the DNA unit fragments. The linear approximation curve obtained from (2) having higher accuracy has a general formula of f (N) =0.0058 ^* CV(％) ^* exp(-0.0058 ^* CV(％) ^* N). In addition, as can be seen from fig. 21, λ =0.0058 ^* CV (%) exhibited a square value of the correlation coefficient as high as 0.99, being a high correlation. Therefore, it was confirmed that this general formula had no problem.

< qualitative analysis of reaction Rate of ligation >

In the connection simulation described above, it is assumed that all connections between normal combinations of protrusions are completed. In order to investigate the approximation degree between the ligation reaction conditions in the actual gene assembly and the simulated reaction conditions, the kinetics of ligation reaction (kinetics) was examined.

First, in a ligation reaction performed under actual gene assembly conditions using lambda phage-reconstructed 51-fragment ligation, ligation products were sampled at various reaction times, and all 51 ligation sites were qualitatively analyzed to investigate the extent to which ligation actually proceeded. The average concentration of the unit DNA fragments used for ligation was about 0.2 fmol/. Mu.l, T4DNA ligase was added to the solution of the unit DNA fragments, reaction was performed at 37 ℃ for 0, 1.25, 2.5, 5, 10, 20, 40, 80, 160, and 320 minutes, and then a part of each reaction solution was collected, a primer set for quantitative PCR designed to amplify DNA spanning the junction of the 2 regularly ligated unit DNA fragments and a primer set for quantitative PCR designed to amplify only the inside of each unit DNA fragment were used, commercially available lambda phage genomic DNA (dongyo) or an assembled plasmid constructed was cleaved with restriction enzymes, and the degree of progress of ligation of each fragment was examined using a dilution series of linearized DNA as an index. As a result, it was confirmed that the ligation was completed in about 10 minutes at any of the junctions under the present reaction conditions, and the ligation was almost completely completed in a reaction time of actually 4 hours (240 minutes). After a sufficient time (after 40 minutes), the reaction degree of almost all the ligation sites was present at a ratio of about 1 to the value obtained based on the smaller number of fragments in each unit DNA fragment, and it was confirmed that almost all the ligated DNA fragments were ligated to the correct target.

< estimation of erroneous connection ratio >

In order to confirm the ligation state in more detail, in the assemblies obtained in the above experiments for reconstructing the lambda phage genome, the nucleotide sequences were determined for all clones (# 1, #2, #5, #7, #8, #9, #10, # 11) except for #3, #4, #6, #12 for which the entire nucleotide sequences were completely determined, and thus the positions ligated in the wrong combinations were determined. The misconnection sites for each clone are shown in FIG. 22. From the results, it was confirmed that 1 or 2 misconnections were present in the interior of the 7 clones except for the #11 clone, and all the misconnections were determined. On the other hand, in the clone #11, the identical unit DNA fragments were found to be present repeatedly in the assembled DNA, and although complete structural confirmation could not be carried out, it was found that 6 mis-ligated sites coexisted. The detailed number of unit DNA fragments of #11 was not specified, and therefore, the occurrence frequency of erroneous ligation was calculated for all clones except #11, and as a result, it was confirmed that erroneous ligation existed at a rate of 1 out of about 46 junctions, that is, erroneous ligation occurred at a low rate of about 2.2%. This result is not contradictory to the above-mentioned results of quantitative PCR.

< verification of the correspondence between the size distribution of the actual ligation products and the simulation >

Based on the 2 verifications of the estimation of the ratio of erroneous connection and the qualitative analysis of the reaction rate of connection, it is estimated that almost all of the connection is completed after a sufficient time of 4 hours has elapsed in the actual connection reaction, and the probability of occurrence of erroneous connection is small. Then, it was verified by the following method whether the size distribution of the actual ligation product can be predicted by simulation for the actual unit DNA fragment group having a difference in concentration between the initial unit DNA fragments.

First, as a material, a population for which a difference CV of 7.5% in the unit DNA fragment concentration was observed by quantitative PCR for the lambda phage genome reconstruction experiment was used. The observed value of the quantitative PCR includes a measurement error with a value of CV =3.6%, and thus, it is assumed that the CV of the difference of the real unit DNA fragments may be lower than CV =7.5%. Therefore, if the CV that can be a true value is obtained by simulation, and as a result, if the true CV is estimated to be 6.6%, the measurement error CV =3.6%, the observed value can be CV =7.5%. Then, assuming that the difference between the true unit DNA fragments of the group is CV =6.6%, 640 fragments are generated by the RAND () method on average, and a simulation of concatenation is performed for 51 initial unit DNA fragments satisfying the condition of CV = 6.6%. In this simulation, except for m representing a reaction ratio of 100% _min Also, 100 independent random number groups were prepared for m values at the time of connection at connection efficiencies of 95%, 96%, 97%, 98%, and 99%, respectively, and the reaction was continued until the respective designated m values were reached. The distribution of the obtained ligation products of 100 clusters was integrated, and the length of the DNA of each mock ligation product of the unit DNA fragment was determined in bp units using the parameters of F (N, L, R). Next, the unit DNA fragment group in the actual lambda phage genome reconstitution experiment, in which the difference in the number of fragments was CV =6.6%, was reacted at 37 ℃ for 4 hours as described in the above "qualitative analysis of ligation reaction rate", and after the reaction, the DNA was electrophoresed for 16 hours under the electrophoresis conditions of 0.5 × TBE, 5V/cm, and 30sec cycles using a CHEF type pulsed field gel electrophoresis apparatus (manufactured by BIO CRAFT), to examine the actual molecular weight distribution of the DNA. The photograph of the electrophoresis is shown in FIG. 23. The DNA density distribution of the electrophotograph obtained by NIHimage software was superimposed on the expected DNA distribution map of each ligation efficiency obtained by simulation and compared. The results are shown in FIG. 24. From fig. 24, it was confirmed that when the ligation efficiency was 98% to 100%, the molecular weight distribution of the DNA obtained by electrophoresis approximately matched the expected DNA distribution map, and in particular, the molecular weight having the maximum value of the maximum concentration well matched the result of the ligation efficiency of 98%. It follows that the results of the simulation canIt was possible to substantially completely reproduce the case where almost all the ligation was completed by the 4-hour ligation reaction and the erroneous ligation occurred at a rate of about 2%.

< generalization of connection simulation >

In practice, the concentration difference of the unit DNA fragments to be assembled cannot be avoided, and in order to summarize the level to which the concentration difference of the unit DNA fragments must be actually controlled, the general formula f (N) =0.0058 obtained as described above ^* CV(％) ^* exp(-0.0058 ^* CV(％) ^* N) is plotted, shown in fig. 25. In the current gene assembly experiment, the difference in DNA concentration was about CV (%) =6.6, and as can be seen from fig. 25, when CV (%) =6.6, about 40% of the unit DNA fragments used in assembly of 51 fragments were integrated into a ligation product having an r value greater than 1. Further, as is clear from fig. 25, if a new gene assembly is planned using a group of 102 fragments, which is a 2-fold assembly scale, CV (%) =3.3 needs to be achieved if the same assembly efficiency as 51 fragments is expected. In addition, the general formula f (N) =0.0058 is used ^* CV(％) ^* exp(-0.0058 ^* CV(％) ^* N), the relationship between the difference in the concentration of the unit DNA fragments and the average number of unit DNA fragments of 1 ligation product was determined. The results are shown in FIG. 26. It can be seen that by using the general formula f (N) =0.0058 ^* CV(％) ^* exp(-0.0058 ^* CV(％) ^* N), it can be easily estimated from the CV (%) value that the average unit DNA fragment content of 1 ligation product is present.

Claims (9)

1. A method of preparing a unitary DNA composition, the method comprising the steps of:

(1) Preparing a solution containing each of a plurality of unit DNAs to which an additional sequence is ligated;

(2) Preparing each solution, measuring the concentration of the unit DNA in each solution in a state where the unit DNA is linked with an additional sequence, and sorting each solution based on the result of the measurement so that the number of moles of the unit DNA in each solution is approximately the same; and

(3) And (3) cleaving and separating the additional sequence and the unit DNA by restriction enzyme treatment after the step (2).

2. The method for preparing a unit DNA composition according to claim 1, wherein the step (3) is a step of preparing a mixture containing the plurality of unit DNAs cleaved by cleavage and separation of the addition sequence and the unit DNA by restriction enzyme treatment;

the method further comprises the following steps:

(4) And (4) a step of removing the additional sequence from the mixture solution after the step (3) to obtain the unit DNA.

3. The method for preparing a unit DNA composition according to claim 1,

the plurality of unit DNAs comprising the same kind of unit DNA groups as the restriction enzymes used for the removal of the additional sequences,

the step (3) is a step of subjecting the mixture containing the unit DNA set to a restriction enzyme treatment.

4. The method for producing a unit DNA composition according to claim 1, wherein the unit DNA to which the additional sequence is ligated has a circular structure, and the additional sequence is a plasmid DNA sequence having an origin of replication.

5. A method of preparing a unitary DNA composition, the method comprising the steps of:

(1) Preparing a solution containing each of a plurality of unit DNAs to which an additional sequence is ligated;

(2) Preparing each solution, measuring the concentration of the unit DNA in each solution in a state where the unit DNA is linked with an additional sequence, and sorting each solution based on the result of the measurement so that the number of moles of the unit DNA in each solution is approximately the same; and

(3) A step of cleaving and separating the additional sequence and the unit DNA by a restriction enzyme treatment after the step (2),

wherein the standard deviation of the distribution of the total length of the base lengths of the unit DNAs and the total base length of the additional sequences linked to the unit DNAs is within. + -. 20% of the average total length.

6. A method of preparing a unitary DNA composition, the method comprising the steps of:

(1) Preparing a solution containing each of a plurality of unit DNAs to which an additional sequence is ligated;

(2) Preparing each solution, measuring the concentration of the unit DNA in each solution in a state where the unit DNA is linked with an additional sequence, and sorting each solution based on the result of the measurement so that the number of moles of the unit DNA in each solution is approximately the same; and

(3) A step of cleaving and separating the addition sequence and the unit DNA by a restriction enzyme treatment after the step (2),

wherein the average base length of the additional sequence linked to each of the unit DNAs is 2 times or more the average base length of the unit DNA.

7. The method for producing a unit DNA composition according to any one of claims 1 to 6, wherein each of the unit DNAs has a length of 1600bp or less.

8. A method of preparing a unitary DNA composition, the method comprising the steps of:

(1) Preparing a solution containing each of a plurality of unit DNAs to which an additional sequence is ligated;

(2) After preparing each solution, measuring the concentration of the unit DNA in each solution in a state where the additional sequence is linked to the unit DNA, and sorting each solution based on the result of measuring the concentration of the unit DNA in each solution so that the number of moles of the unit DNA in each solution is approximately the same; and

(3) A step of cleaving and separating the addition sequence and the unit DNA by a restriction enzyme treatment after the step (2),

wherein the unit DNA is used for preparing a DNA link containing an assembly DNA composed of the unit DNA,

in the method, the step of preparing a solution containing the unit DNA includes the steps of:

the unit DNA having a non-palindromic sequence at an end is designed so as to be bounded by non-palindromic sequences in the assembled DNA in the vicinity of the sequence at positions where the assembled DNA is divided into equal base lengths when the base lengths of the sequences of the assembled DNA are divided by the number of types of the unit DNA.

9. A method for producing a DNA construct for use in microbial cell transformation, said DNA construct comprising more than 1 assembled DNA unit comprising a vector DNA and an assembled DNA, said vector DNA comprising an origin of replication effective in a host microorganism, said method comprising the steps of:

a step of preparing a unit DNA composition by the method according to any one of claims 1 to 6 and 8;

preparing the vector DNA;

removing each additional sequence from the prepared unit DNA having the additional sequence ligated to the solution using a restriction enzyme; and

a step of linking the vector DNA and the unit DNAs to each other after the removal step,

wherein the vector DNA and the unit DNAs have a structure capable of being repeatedly linked in a state of maintaining the order of each other,

the assembly DNA includes DNAs in which the unit DNAs are linked to each other.

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